EP4255487A1 - Polypeptides de détection et de traitement d'infection à coronavirus - Google Patents

Polypeptides de détection et de traitement d'infection à coronavirus

Info

Publication number
EP4255487A1
EP4255487A1 EP21901659.9A EP21901659A EP4255487A1 EP 4255487 A1 EP4255487 A1 EP 4255487A1 EP 21901659 A EP21901659 A EP 21901659A EP 4255487 A1 EP4255487 A1 EP 4255487A1
Authority
EP
European Patent Office
Prior art keywords
antibody
amino acid
polypeptide
antigen binding
variable region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21901659.9A
Other languages
German (de)
English (en)
Inventor
Patrick Wilson
Haley Dugan
Christopher Stamper
Yoshihiro Kawaoka
Peter Halfmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Chicago
Wisconsin Alumni Research Foundation
Original Assignee
University of Chicago
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Chicago, Wisconsin Alumni Research Foundation filed Critical University of Chicago
Publication of EP4255487A1 publication Critical patent/EP4255487A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • aspects of the invention relate to at least the fields of virology and molecular biology.
  • the inventors have comprehensively characterized the SARS-CoV-2-specific B cell repertoire in convalescent COVID-19 patients and generated mAbs against the spike, ORF8, and NP proteins. Together, the inventors’ data reveal key insights into antigen specificity and B cell subset distribution upon SARS-CoV-2 infection in the context of age, sex, and disease severity. Aspects of the disclosure relate to novel antibody and antigen binding fragments, as well as methods of using these fragments. Further aspects relate to polypeptides comprising the antigen binding fragment(s) of the disclosure, and compositions comprising the polypeptides, antibodies, and/or antigen binding fragments of the disclosure.
  • nucleic acids encoding an antibody or antigen binding fragment of the disclosure also relates to nucleic acids encoding an antibody heavy chain, wherein the nucleic acid has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOS: 1621-1710 or 2707-2755.
  • nucleic acids encoding an antibody light chain of the disclosure, wherein the nucleic acid has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOS: 1711-1800 or 2756-2804.
  • nucleic acids of the disclosure may be DNA or RNA.
  • a method of a making a cell comprising transferring one or more nucleic acid(s) of the disclosure into a cell.
  • the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.
  • the method further comprising isolating the expressed polypeptide.
  • the cell may be further defined as a human cell, B cell, T cell, Chinese hamster ovary, NSO murine myeloma cell, PER.C6 cell, or a cell described herein.
  • Further aspects of the disclosure relate to a method for treating or preventing a coronavirus infection in a subject, the method comprising administering to the subject an antibody, antigen binding fragment, polypeptide, nucleic acid, or host cell of the disclosure. Yet further aspects relate to a method for evaluating a sample from a subject, the method comprising contacting a biological sample from the subj ect, or extract thereof, with at least one antibody, antigen binding fragment, or polypeptide of the disclosure. Also disclosed is a method for diagnosing a SARS-CoV-2 infection in a subject, the method comprising contacting a biological sample from the subject, or extract thereof, with at least one antibody, antigen binding fragment, or polypeptide of any one of the disclosure.
  • compositions of the disclosure are formulated as a vaccine for the treatment or prevention of a coronoavirus infection.
  • the antibodies, antigen binding fragments, or compositions of the disclosure are used in a vaccine for preventing coronaviral infections in a subject that does not have a coronaviral infection.
  • the antibodies, antigen binding fragments, or compositions of the disclosure are used to treat a subject having a coronaviral infection.
  • a method of a making a cell comprising transferring one or more nucleic acid(s) of the disclosure into a cell.
  • the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.
  • the method further comprising isolating the expressed polypeptide.
  • Aspects describe a method for producing a polypeptide comprising transferring one or more nucleic acid(s) or vector(s) of the disclosure into a cell and isolating polypeptides expressed from the nucleic acid.
  • the cell may be further defined as a human cell, B cell, T cell, Chinese hamster ovary, NSO murine myeloma cell, PER.C6 cell, or a cell described herein.
  • Further aspects of the disclosure relate to a method for treating or preventing a coronavirus infection in a subject, the method comprising administering to the subject an antibody, antigen binding fragment, polypeptide, nucleic acid, or host cell of the disclosure. Yet further aspects relate to a method for evaluating a sample from a subject, the method comprising contacting a biological sample from the subj ect, or extract thereof, with at least one antibody, antigen binding fragment, or polypeptide of the disclosure. Also disclosed is a method for diagnosing a SARS-CoV-2 infection in a subject, the method comprising contacting a biological sample from the subject, or extract thereof, with at least one antibody, antigen binding fragment, or polypeptide of any one of the disclosure.
  • compositions of the disclosure are formulated as a vaccine for the treatment or prevention of a coronoavirus infection.
  • the antibodies, antigen binding fragments, or compositions of the disclosure are used in a vaccine for preventing coronaviral infections in a subject that does not have a coronaviral infection.
  • the antibodies, antigen binding fragments, or compositions of the disclosure are used to treat a subject having a coronavirus infection.
  • aspects of the disclosure relate to an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1.
  • an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having or having at least 80% sequence identity or having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity with a HCDR1, HCDR2, and HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having or having at least least 80% sequence identity or having or
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 may be determined from the variable region sequences by methods known in the art.
  • the CDR is HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 determined by the Chothia method.
  • the CDR is HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 determined by the Kabat method.
  • the CDR is HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 determined by the IMGT method.
  • aspects of the disclosure relate to an antibody or antigen binding fragment in which the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence that has at least 80% sequence identity to an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone
  • the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95
  • the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequence of an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone.
  • an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the HCDR1, HCR2, HCR3 from a heavy chain variable region of a antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having or having at least 60, 61, 62, 63, 64, 65,
  • the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having or having at least 80% sequence identity to the HCDR1, HCR2, HCR3 from a heavy chain variable region of a antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.
  • the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having the amino acid sequence of an of a HCDR1, HCDR2, and HCDR3 of a clone of Table 1 and the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 comprising the amino acid sequence of the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1
  • the polypeptides of the disclosure may comprise at least two antigen binding fragments, wherein each antigen binding fragment is independently selected from an antigen binding fragment of the disclosure.
  • the polypeptide is multivalent. In some aspects, the polypeptide is multispecific.
  • the polypeptide is bispecific. In some aspects, the polypeptide comprises, comprises at least, or comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen binding regions. Each antigen binding region may be independently selected from an antigen binding region of the disclosure. In some aspects, the polypeptide may have repeated units of the same antigen binding region, such as at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeated units.
  • the heavy chain variable region comprises an amino acid sequence with at least 80% sequence identity to a heavy chain variable region of an antibody clone of Table 1 and/or the light chain variable region comprises an amino acid sequence with at least 80% sequence identity to the light chain variable region of the same antibody clone of Table 1.
  • the heavy chain variable region comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to a heavy chain variable region of an antibody clone of Table 1 and/or the light chain variable region comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88
  • the antibody or antigen binding fragment comprises a heavy chain framework region (HFR) 1, HFR2, HFR3, and HFR4 and light chain framework region (LFR) 1, LFR2, LFR3, and LFR4, and wherein the HFR1, HFR2, HFR3, and HFR4 comprises an amino acid sequence with at least 80% sequence identity to an HFR1 , HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1, and the LFR1, LFR2, LFR3, and LFR4 comprises an amino acid sequence with at least 80% sequence identity to the LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone of Table 1.
  • HFR heavy chain framework region
  • LFR3 and HFR4 comprises an amino acid sequence with at least 80% sequence identity to an HFR1 , HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1
  • the LFR1, LFR2, LFR3, and LFR4 comprises an amino acid sequence with at least 80% sequence identity to the LFR1, L
  • the antibody or antigen binding fragment comprises a heavy chain framework region (HFR) 1, HFR2, HFR3, and HFR4 and light chain framework region (LFR) 1, LFR2, LFR3, and LFR4, and wherein the HFR1, HFR2, HFR3, and HFR4 comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to an HFR1, HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1, and the LFR1, LFR2, LFR3, and LFR4 comprises an amino acid sequence having or having at least
  • the HFR1, HFR2, HFR3, and HFR4 comprises the amino acid sequence of an HFR1, HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1
  • the LFR1, LFR2, LFR3, and LFR4 comprises the amino acid sequence of the LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone of Table 1.
  • the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence with at least 70% sequence identity to a heavy chain of an antibody clone of Table 1 and the light chain comprises an amino acid sequence with at least 70% sequence identity to the light chain of the same antibody clone of Table 1.
  • the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identityto a heavy chain of an antibody clone of Table 1 and the light chain comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
  • the heavy chain variable region comprises a heavy chain framework region that has or has at least 80% sequence identity to a heavy chain framework region of an antibody clone of Table 1 and the light chain variable region comprises a light chain framework region that has or has at least 80% sequence identity to a light chain framework region of the same antibody clone of Table 1.
  • the heavy chain variable region comprises a heavy chain framework region having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to a heavy chain framework region of an antibody clone of Table 1 and the light chain variable region comprises a light chain framework region having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
  • the heavy chain variable region comprises at least 70% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 70% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1.
  • the heavy chain variable region comprises at least 75% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 75% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1.
  • the heavy chain variable region comprises at least 80% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 80% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1
  • the heavy chain variable region comprises at least 85% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 85% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1.
  • the heavy chain variable region comprises at least 90% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 90% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1.
  • the heavy chain variable region comprises at least 95% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 95% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1.
  • the antibody or antigen binding fragment of the disclosure may be human, chimeric, or humanized.
  • the antibody, or antigen binding fragment binds a SARS-CoV-2 Spike, NP protein, or ORF8 with a kD of about 10' 6 nM to about 10" 12 pM.
  • the antibody, or antigen binding fragment binds a SARS-CoV-2 Spike, NP protein, or ORF8 with a kD of about, a kD of at least, or a kD of at most 10' 3 , 10' 4 , 10' 5 , 10' 6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 , 10 -17 , or 10 -18 (or any derivable range therein) ⁇ M, nM, or pM.
  • the antibody or antigen binding fragment specifically binds to a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.
  • the antibody may be further defined as a neutralizing antibody.
  • the antibody or antigen binding fragment is further defined as a human antibody or antigen binding fragment, humanized antibody or antigen binding fragment, recombinant antibody or antigen binding fragment, chimeric antibody or antigen binding fragment, an antibody or antigen binding fragment derivative, a veneered antibody or antigen binding fragment, a diabody, a monoclonal antibody or antigen binding fragment, a single domain antibody, or a single chain antibody.
  • the antigen binding fragment is further defined as a single chain variable fragment (scFv), F(ab’)2, Fab’, Fab, Fv, or rlgG.
  • the antibody, antigen binding fragment, or polypeptide is operatively linked to a detectable label. Detectable labels are described herein.
  • aspects of the disclosure also relate to multi-specific and/or multivalent antibodies and polypeptides. Accordingly, aspects relate to bivalent or bispecific antibodies that comprise two antigen binding fragments, wherein the antigen binding fragment is two of the same antigen binding fragments or two different antigen binding fragments described herein.
  • the disclosure also provides for multi-specific polypeptides. Aspects relate to polypeptides comprising or comprising at least 2, 3, 4, 5, or 6 antigen binding fragments.
  • the antigen binding fragment may be at least 2, 3, 4, 5, or 6 scFv, F(ab’)2, Fab’, Fab, Fv, or rlgG, or combinations thereof.
  • the polypeptide and/or antigen binding fragments of the disclosure may comprise a linker between a heavy chain and light chain variable region or between antigen binding fragments.
  • the linker may be a flexible linker.
  • Exemplary flexible linkers include glycine polymers (G)n, glycine- serine polymers (including, for example, (GS)n, (GSGGS-SEQ ID NO:2805)n, (G4S)n and (GGGS-SEQ ID NO:2806)n, where n is an integer of at least one.
  • n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein).
  • Exemplary linkers can comprise or consist of GGSG (SEQ ID NO:2807), GGSGG (SEQ ID NO:2808), GSGSG(SEQ ID NO:2809), GSGGG (SEQ ID NO:2810), GGGSG (SEQ ID NO:2811), GSSSG (SEQ ID NO:2812), and the like.
  • the coronavirus infection is a SARS-CoV-2 infection. In some aspects, the coronavirus infection is a SARS-CoV infection. In some aspects, the coronavirus infection is a MERS-CoV infection. In some aspects, the coronavirus infection is a HCoV- OC43, HCoV-HKUl, HCoV-229E, or HCoV-NL63 infection.
  • compositions of the disclosure may comprise a pharmaceutical excipient, carrier, or molecule described herein.
  • the composition further comprises an adjuvant or an immunostimulator.
  • adjuvants or immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+M
  • compositions may comprise more than one antibody and/or antigen binding fragment of the disclosure. Accordingly, compositions of the disclosure may comprise, may comprise at least, or may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antibodies and/or antigen binding fragments of the disclosure.
  • the compositions of the disclosure may be formulated for a route of administration described herein.
  • compositions are formulated for parenteral, intravenous, subcutaneous, intramuscular, or intranasal administration.
  • the compositions is formulated for intranasal administration.
  • the host cell is a human cell, B cell, T cell, Chinese hamster ovary, NS0 murine myeloma cell, or PER.C6 cell. In some aspects, the host cell is a cell type or cell population described herein.
  • the subject or patient may be a human subject or a human patient.
  • the subject or patient is a non-human animal.
  • the non-human animal is a bat, monkey, camel, rat, mouse, rabbit, goat, chicken, bird, cat, or dog.
  • the subject may further be defined as an at-risk subject.
  • At-risk subjects include health care workers, immunocompromised subjects, people over the age of 65, or those with at least one or at least two underlying conditions.
  • Example of underlying conditions include obesity, high blood pressure, autoimmunity, cancer, and asthma.
  • the subject has one or more symptoms of a coronavirus infection.
  • Symptoms of a coronavirus infection include, but are not limited to elevated temperature or a fever of 100.0°F or more, loss of taste or smell, cough, difficulty breathing, shortness of breath, fatigue, headache, chills, sore throat, congestion or runny nose, shaking or exaggerated shivering, significant muscle pain or ache, diarrhea, and/or nausea or vomiting.
  • the subject does not have any symptoms of a coronavirus infection
  • the subject has been diagnosed with a coronavirus infection.
  • the subject has not been diagnosed with a coronavirus infection.
  • the subject has been previously treated for a coronavirus infection.
  • the subject has been previously vaccinated for coronavirus.
  • the subject has not been previously vaccinated for coronavirus.
  • the previous treatment comprises a pain reliever, such as acetaminophen or ibuprofen, a steroid such as dexamethasone, prednisolone, beclomethasone, fluticasone, or methylprednisone or an antiviral such as remdesivir.
  • the subject is administered an additional therapeutic.
  • the additional therapeutic may comprise one or more of a pain reliever, such as acetaminophen or ibuprofen, a steroid such as dexamethasone, prednisolone, beclomethasone, fluticasone, or methylprednisone or an antiviral such as remdesivir.
  • a pain reliever such as acetaminophen or ibuprofen
  • a steroid such as dexamethasone, prednisolone, beclomethasone, fluticasone, or methylprednisone or an antiviral such as remdesivir.
  • the additional therapeutic comprises dexamethasone.
  • the additional therapeutic comprises remdesivir.
  • the method further comprises incubating the antibody, antigen binding fragment, or polypeptide under conditions that allow for the binding of the antibody, antigen binding fragment, or polypeptide to antigens in the biological sample or extract thereof.
  • the method further comprises detecting the binding of an antigen to the antibody, antigen binding fragment, or polypeptide.
  • the method further comprises contacting the biological sample with at least one capture antibody, antigen, or polypeptide
  • the at least one capture antibody, antigen binding fragment, or polypeptide may be an antibody, polypeptide, or antigen binding fragment of the disclosure.
  • the capture antibody is linked or operatively linked to a solid support.
  • operatively linked refers to a situation where two components are combined or capable of combining to form a complex.
  • the components may be covalently attached and/or on the same polypeptide, such as in a fusion protein or the components may have a certain degree of binding affinity for each other, such as a binding affinity that occurs through van der Waals forces.
  • the biological sample comprises a blood sample, urine sample, fecal sample, or nasopharyngeal sample
  • the at least one antibody, antigen binding fragment, or polypeptide may be operatively linked to a detectable label.
  • the method further comprises incubating the antibody, antigen binding fragment, or polypeptide under conditions that allow for the binding of the antibody, antigen binding fragment, or polypeptide to antigens in the biological sample or extract thereof. In some aspects, the method further comprises detecting the binding of an antigen to the antibody, antigen binding fragment, or polypeptide. In some aspects, the method further comprises contacting the biological sample with at least one capture antibody, antigen, or polypeptide. In some aspects, the biological sample comprises a blood sample, urine sample, fecal sample, or nasopharyngeal sample.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:3-5, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 12- 14, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:21-23, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:30- 32, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:39-41, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:48- 50, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:57-59, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:66- 68, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:75-77, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 84- 86, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:93-95, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 102- 104, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:111-113, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:120-122, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 129-131, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:138-140, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:147-149, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:156-158, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:165-167, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:174-176, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 183 -185, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:192-194, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :201-203, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:210-212, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :219-221, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:228-230, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:237-239, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:246-248, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:255-257, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:264-266, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:273-275, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:282-284, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :291-293, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:300-302, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:309-311, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:318-320, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:327-329, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:336-338, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:345-347, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:354-356, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:363-365, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:372-374, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:381-383, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:390-392, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :399-401, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:408-410, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :417-419, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:426-428, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :435-437, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:444-446, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:453-455, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:462-464, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :471-473, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:480-482, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:489-491, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NQS:498-500, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:507-509, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:516-518, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:525-527, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:534-536, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:543-545, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:552-554, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :561-563, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:570-572, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:579-581, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:588-590, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:597-599, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:606-608, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :615-617, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:624-626, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:633-635, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:642-644, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :651-653, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:660-662, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:669-671, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:678-680, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:687-689, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:696-698, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:705-707, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:714-716, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:723-725, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:732-734, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :741-743, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:750-752, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:759-761, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:768-770, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:777-779, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:786-788, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:795-797, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NQS:804-806, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:813-815, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 822-824, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:831-833, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:840-842, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:849-851, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:858-860, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:867-869, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:876-878, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:885-887, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:894-896, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NQS:903-905, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:912-914, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :921-923, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:930-932, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS :939-941, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:948-950, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:957-959, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:966-968, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:975-977, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:984-986, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:993-995, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1002-1004, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1011-1013, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1020-1022, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1029-1031, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1038-1040, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1047-1049, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1056-1058, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1065-1067, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1074-1076, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1083-1085, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1092-1094, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1101-1103, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1110-1112, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1119-1121, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1128-1130, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1137-1139, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1146-1148, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1155-1157, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1164-1166, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1173-1175, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1182-1184, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1191-1193, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1200-1202, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1209-1211, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1218-1220, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1227-1229, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1236-1238, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1245-1247, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1254-1256, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1263-1265, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1272-1274, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1281-1283, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1290-1292, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1299-1301, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1308-1310, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1317-1319, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1326-1328, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1335-1337, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1344-1346, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1353-1355, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1362-1364, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1371-1373, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1380-1382, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1389-1391, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1398-1400, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1407-1409, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1416-1418, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1425-1427, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1434-1436, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1443-1445, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1452-1454, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1461-1463, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1470-1472, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1479-1481, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1488-1490, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1497-1499, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1506-1508, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1515-1517, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1524- 1526, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1533-1535, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1542- 1544, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1551-1553, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1560-1562, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1569-1571, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1578-1580, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1587-1589, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1596-1598, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1605-1607, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1614-1616, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1827-1829, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1836- 1838, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1845-1847, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:1854-1856, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1863-1865, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1872- 1874, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1881-1883, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1890- 1892, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1899-1901, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1908-1910, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1917-1919, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1926-1928, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1935-1937, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1944-1946, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1953-1955, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1962-1964, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1971-1973, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1980-1982, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1989-1991, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS: 1998-2000, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2007-2009, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2016-2018, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2025-2027, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2034-2036, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2043-2045, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2052-2054, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2061-2063, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2070-2072, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2079-2081, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2088-2090, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2097-2099, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2106-2108, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2115-2117, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2124-2126, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2133-2135, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2142-2144, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2151-2153, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2160-2162, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2169-2171, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2178-2180, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2187-2189, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2196-2198, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2205-2207, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2214-2216, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2223-2225, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2232-2234, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2241-2243, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2250-2252, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2259-2261, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2268-2270, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2277-2279, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2286-2288, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2295-2297, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2304-2306, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2313-2315, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2322-2324, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2331-2333, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2340-2342, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2349-2351, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2358-2360, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2367-2369, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2376-2378, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2385-2387, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2394-2396, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2403-2405, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2412-2414, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2421-2423, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS :2430-2432, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2439-2441, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2448-2450, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2457-2459, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2466-2468, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2475-2477, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2484-2486, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2493-2495, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2502-2504, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2511-2513, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2520-2522, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2529-2531, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2538-2540, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2547-2549, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2556-2558, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2565-2567, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2574-2576, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2583-2585, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2592-2594, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2601-2603, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2610-2612, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2619-2621, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2628-2630, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2637-2639, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2646-2648, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2655-2657, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2664-2666, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2673-2675, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2682-2684, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1 , HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:2691-2693, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:2700-2702, respectively.
  • aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region and a light chain variable region of SEQ ID NOS:2 and 11, SEQ ID NOS :20 and 29, SEQ ID NOS:38 and 47, SEQ ID NOS:56 and 65, SEQ ID NOS:74 and 83, SEQ ID NOS:92 and 101, SEQ ID NOS:110 and 119, SEQ ID NOS:128 and 137, SEQ ID NOS: 146 and 155, SEQ ID NOS: 164 and 173, SEQ ID NOS: 182 and 191, SEQ ID NOS:200 and 209, SEQ ID NOS:218 and 227, SEQ ID NOS:236 and 245, SEQ ID NOS:254 and 263, SEQ ID NOS:272 and 281, SEQ ID NOS:290 and 299, SEQ ID NOS:308 and 317, SEQ ID NOS:326 and 335, SEQ ID NOS:3
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that embodiments described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”
  • “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
  • any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.
  • any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
  • FIG. la-g B cell subsets enriched for SARS-CoV-2-reactivity are revealed by transcriptome, Ig repertoire, and probe binding
  • a Model demonstrating antigen probe preparation and representative gating strategy for sorting antigen-positive B cells
  • c Integrated transcriptional UMAP analysis of distinct B cell clusters and the corresponding number of B cells per cluster
  • d Feature library enrichment of antigen-probe-positive B cells by cluster
  • e Percent probe reactivity of all B cells per cluster
  • f Ig isotype usage and VH gene SHM for all antigen-positive B cells per cluster Bars indicate median with interquartile range
  • g Representative visualization of antigen reactivity revealing antigen-specific B cells. Axes indicate antigen probe intensities.
  • FIG. 2a-d Transcriptional analysis distinguishes naive, innate-like and MBC subsets specific to SARS-CoV-2 proteins, a-b, Trajectory (a) and pseudotime (b) analyses of clusters 0-11 reveals least to most differentiated clusters.
  • Cluster 12 is excluded from trajectory analysis as it represents a separate partition as defined by Monocle3.
  • c Heatmap showing the top twenty most differentially expressed genes per cluster. Red stars denote genes used in memory B cell (MBC) identification, d, Volcano plots comparing differentially expressed genes in MBC-like clusters relative to cluster 2 (naive B cells).
  • Genes used in MBC identification are indicated: cd27, cd38, hhex, zeb2, pou2afl , spib, cd80, cd86.
  • mcll, prdml, abpl, manf, bach2, pax5 Red-colored dots represent a log fold change in expression >0. 1 and an adj-p value ⁇ 0.01.
  • Putative B cell subset identities are highlighted where they could be clearly defined (a).
  • FIG. 3a-p SARS-CoV-2 -reactive B cells exhibit unique features for isotype, SHM, subset of origin, and VH gene usage, a-1, Ig isotype, VH gene SHM, and distribution of B cells by integrated cluster for spike- (a, b, c, d), NP- (e, f, g, h) and ORF8-specific B cells (i, j, k, 1).
  • m-p Tree maps showing frequency of VH gene locus usage for total spike (including RBD) (m), RBD only (n), NP (o), and ORF8-specific B cells (p). Numbers in the center of each pie chart and below each tree map indicate number of cells analyzed per reactivity.
  • ASCs IgG/IgA antibody secreting cells
  • Percentage of antigen-probe-positive cells by subject d, Percentage of antigen-probe-positive cells stratified by age (years), sex, and symptom duration (weeks)
  • e Two-sided spearman correlation between percentage of all cells specific to ORF8 and subject age with p and r values indicated
  • f Percentage of antigen-probe- positive B cells in MBC-like clusters (3, 4, 5, 6, 7, 9, and 12) or naive and innate-like clusters (0, 1, 2, 8, 10, 11) stratified by age, sex, and symptom duration
  • VH gene SHM for antigen-specific cells from a given age (g), sex (h), or symptom duration group (i).
  • FIG. 6a-c Additional characteristics of B cells comprising integrated clusters, a, Antigen-probe-positive B cell distribution across integrated clusters by subject with the number of cells per subject indicated, b, Variable gene segment usage in B cell receptor heavy chains of antigen-probe-positive B cells across integrated clusters, c, Diagrams showing antigen- probe-positive B cells per cluster with probe intensities for the indicated antigens plotted on the axes.
  • FIG. 7 Expression of MBC and LLPC gene markers in integrated clusters. Normalized expression levels of the indicated genes represented as violin plots.
  • FIG. 8a-i Heavy and light chain features of SARS-CoV-2 reactive B cells, a-b, Heavy chain (HC, a) and light chain (LC, b) complementarity determining region 3 (CDR3) lengths, shown by antigen-reactivity, c-d, HC (c) and LC (d) isoelectric points pl, shown by antigen-reactivity, e, Number of light chain (LC) somatic hypermutations (SHM), shown by antigen-reactivity, f-i.
  • CDR3 complementarity determining region 3
  • Whole spike antigen probe intensities are plotted for RBD-binding mAbs.
  • Fig. 1 la-c B cell receptor and transcriptional analysis reveals cluster identities, (a) B cell receptor isotype usage, somatic hypermutation (SHM), and antigen reactivity by cluster for all integrated samples. SHM data are plotted with the overlay indicating the median with interquartile range, (b) Heatmap displaying differentially expressed genes across clusters. A summary of cluster identities is provided below, (c) UMAP projections with cell color indicating gene module scoring for the indicated B cell subsets. Also see Tables S5 and S6 [0187] Fig. 12a-j. B cell immunodominance and adaptability landscapes vary in acute infection in convalescence, (a) UMAP projection showing cells colored by time point of blood draw.
  • Fig 13a-f B cells targeting distinct antigens display unique variable gene usages
  • VH variable heavy-chain
  • Fig. 14a-h Neutralization capacity and in vivo protective ability of mAbs to the SARS-CoV-2 spike and intracellular proteins
  • MBCs antigen-specific memory B cells
  • n 0.05145 and ****p ⁇ 0.0001; n.s., not significant,
  • Fig. 16a-d B cell cluster distribution and antigen specificity by subject, Related to Fig. 10.
  • (a-b) Overall cluster distribution (top) and antigen-specificity distribution (bottom) for subjects sorted with SARS2 spike (S), SARS2 RBD, NP, and ORF8 antigens, with (a) or without (b) an endemic HCoV cocktail comprised of S proteins from 229E, NL63, OC43, and HKU1 strains,
  • FIG. 17a-d Expression maps of select genes, Related to Figure 11.
  • (a-d) UMAP projections with cells colored by expression level of indicated genes associated with naive B cells (a), memory B cells b), antibody-secreting cells (c), and mucosal homing (d). Also see Table S6.
  • Fig. 18a-j Further analysis of antigen-specific B cell properties across distinct cohorts and timepoints, Related to Figure 12.
  • Fig. 20a-d Additional analyses of antigen reactivity by clinical parameter, Related to Figure 15.
  • MCC durable memory B cell
  • the inventors identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, the inventors isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8.
  • NP nucleocapsid protein
  • ORF open reading frame
  • B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing.
  • mAbs monoclonal antibodies
  • B cells specific to ORF8 and NP were enriched in naive and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing.
  • the inventors identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration Together, the data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.
  • aspects of the disclosure relate to antibodies, antigen binding fragments thereof, or polypeptides capable of specifically binding to a SARS-CoV-2 spike (S) protein, NP protein, or ORF8. Certain aspects relate to antibodies, or fragments thereof, that specifically bind to a receptor binding domain (RBD) of a SARS-CoV-2 spike protein
  • antibody refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies.
  • antibody or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody.
  • An antigen may possess one or more epitopes that are capable of interacting with different antibodies.
  • epitope includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T-cell receptor.
  • Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics.
  • antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.
  • epitope regions of a given polypeptide can be identified using many different epitope mapping techniques are well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockb erg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc.
  • antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.
  • immunogenic sequence means a molecule that includes an amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host.
  • immunogenic composition means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).
  • an intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains.
  • Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies.
  • the variable or CDR regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human.
  • the antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013).
  • the term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity.
  • a full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL).
  • VL variable region domain
  • CL constant region domain
  • VL fragment means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including CDRs.
  • a VL fragment can further include light chain constant region sequences.
  • the variable region domain of the light chain is at the amino-terminus of the polypeptide.
  • the term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity.
  • a full-length heavy chain has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CHI, CH2, and CH3).
  • VH variable region domain
  • CHI constant region domain
  • CH2 constant region domains
  • VH fragment means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs.
  • a VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype.
  • the VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxy -terminus, with the CH3 being closest to the — COOH end.
  • the isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (p), delta (5), gamma (y), alpha (a), or epsilon (s) chains, respectively.
  • IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4.
  • IgM subtypes include IgMl and IgM2.
  • IgA subtypes include IgAl and IgA2.
  • Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e g., F(ab')2, Fab', Fab, Fv, and the like), including hybrid fragments.
  • An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins.
  • the term “monomer” means an antibody containing only one Ig unit. Monomers are the basic functional units of antibodies.
  • the term “dimer” means an antibody containing two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc, or fragment crystallizable, region). The complex may be stabilized by a joining (J) chain protein.
  • the term “multimer” means an antibody containing more than two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc region). The complex may be stabilized by a joining (J) chain protein.
  • bivalent antibody means an antibody that comprises two antigenbinding sites.
  • the two binding sites may have the same antigen specificities or they may be bi-specific, meaning the two antigen-binding sites have different antigen specificities.
  • Bispecific antibodies are a class of antibodies that have two paratopes with different binding sites for two or more distinct epitopes.
  • bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen.
  • bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies”. Single domain antibodies are sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by a linker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No. W02010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which are specifically incorporated herein by reference in their entirety.
  • Bispecific antibodies can be constructed as: a whole IgG, Fab'2, Fab'PEG, a diabody, or alternatively as scFv. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148: 1547-1553 (1992), each of which are specifically incorporated by reference in their entirety.
  • the antigen-binding domain may be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind a different antigen.
  • the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component.
  • aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells.
  • multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art.
  • diabodies that are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites.
  • the linker functionality is applicable for embodiments of triabodies, tetrabodies, and higher order antibody multimers, (see, e.g, Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2: 1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be advantageous because they can be readily constructed and expressed in E. coli.
  • Diabodies (and other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is kept constant, for instance, with a specificity directed against a protein, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39:167-173, 2017), each of which is hereby incorporated by reference in their entirety.
  • Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities. See, e.g., U.S. Patent No. 6,010,902, incorporated herein by reference in its entirety.
  • the part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope ”
  • the paratope consists of the amino acid residues that make contact with the epitope of an antigen to facilitate antigen recognition.
  • Each of the two Fv fragments of an antibody is composed of the two variable domains, VH and VL, in dimerized configuration.
  • the primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, Framework Regions (FR).
  • the hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal
  • the term hypervariable loop is sometimes used interchangeably with the term “Complementarity Determining Region (CDR).”
  • CDR Complementarity Determining Region
  • the length of the hypervariable loops (or CDRs) varies between antibody molecules.
  • the framework regions of all antibody molecules from a given mammal have high primary sequence similarity /consensus.
  • the consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions.
  • the hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur.
  • CDRs in the VL domain are identified as LI, L2, and L3, with LI occurring at the most distal end and L3 occurring closest to the CL domain.
  • the CDRs may also be given the names CDR-L1, CDR-L2, and CDR-L3.
  • the L3 (CDR-L3) is generally the region of highest variability among all antibody molecules produced by a given organism.
  • the CDRs are regions of the polypeptide chain arranged linearly in the primary structure, and separated from each other by Framework Regions.
  • the amino terminal (N-terminal) end of the VL chain is named FR1 .
  • the region identified as FR2 occurs between LI and L2 hypervariable loops.
  • FR3 occurs between L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain.
  • VH chain which includes three CDRs identified as CDR-H1, CDR-H2 and CDR-H3.
  • the three dimensional, or tertiary, structure of an antibody molecule is such that the framework regions are more internal to the molecule and provide the majority of the structure, with the CDRs on the external surface of the molecule.
  • affinity matured antibodies are enhanced with one or more modifications in one or more CDRs thereof that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s).
  • Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio/Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and/or framework residues employed in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009) in conjugation with computation methods as demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).
  • Chimeric immunoglobulins are the products of fused genes derived from different species; “humanized” chimeras generally have the framework region (FR) from human immunoglobulins and one or more CDRs are from a non-human source.
  • FR framework region
  • portions of the heavy and/or light chain are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • For methods relating to chimeric antibodies see, e g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
  • minimizing the antibody polypeptide sequence from the non-human species optimizes chimeric antibody function and reduces immunogenicity.
  • Specific amino acid residues from non-antigen recognizing regions of the non-human antibody are modified to be homologous to corresponding residues in a human antibody or isotype.
  • One example is the “CDR-grafted” antibody, in which an antibody comprises one or more CDRs from a particular species or belonging to a specific antibody class or subclass, while the remainder of the antibody chain(s) is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass.
  • the V region composed of CDR1, CDR2, and partial CDR3 for both the light and heavy chain variance region from a non-human immunoglobulin are grafted with a human antibody framework region, replacing the naturally occurring antigen receptors of the human antibody with the non-human CDRs.
  • corresponding non-human residues replace framework region residues of the human immunoglobulin.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (F c), typically that of a human immunoglobulin.
  • Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space.
  • Polyclonal antibody preparations typically include different antibodies against different determinants (epitopes).
  • a host such as a rabbit or goat
  • the antigen or antigen fragment generally with an adjuvant and, if necessary, coupled to a carrier.
  • Antibodies to the antigen are subsequently collected from the sera of the host.
  • the polyclonal antibody can be affinity purified against the antigen rendering it monospecific
  • Monoclonal antibodies or “mAb” refer to an antibody obtained from a population of homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.
  • antibody fragments such as antibody fragments that bind to a SARS-CoV-2 spike protein
  • the term functional antibody fragment includes antigenbinding fragments of an antibody that retain the ability to specifically bind to an antigen.
  • VH variable region heavy chain
  • VL variable region heavy chain
  • CHI constant region heavy chain 1
  • CL light chain
  • they lack the Fc region constituted of heavy chain 2 (CH2) and 3 (CH3) domains.
  • Embodiments of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CHI domains; (ii) the Fd fragment type constituted with the VH and CHI domains; (iii) the Fv fragment type constituted with the VH and VL domains; (iv) the single domain fragment type, dAb, (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions.
  • CDR complementarity determining region
  • Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 complementarity determining regions (CDRs) from a light chain variable region. Fusions of CDR-containing sequences to an Fc region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein.
  • CDRs complementarity determining regions
  • Fab fragment means a monovalent antigen-binding fragment of an antibody containing the VL, VH, CL and CHI domains.
  • Fab' fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment.
  • a Fab' fragment includes the VL, VH, CL and CHI domains and all or part of the hinge region.
  • F(ab')2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab' fragments linked by a disulfide bridge at the hinge region.
  • An F(ab')2 fragment includes, for example, all or part of the two VH and VL domains, and can further include all or part of the two CL and CHI domains.
  • Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs.
  • An Fd fragment can further include CHI region sequences.
  • Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CHI domains.
  • the VL and VH include, for example, the CDRs.
  • Single-chain antibodies are Fv molecules in which the VL and VH regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference.
  • (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et al. 1992).
  • the oligomerization domain comprises self-associating a- helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds.
  • (scFv)2 fragments are also known as “miniantibodies” or “minibodies.”
  • a single domain antibody is an antigen-binding fragment containing only a VH or the VL domain.
  • two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody may target the same or different antigens.
  • Fab polypeptides of the disclosure include the Fab antigen binding fragment of an antibody Unless specifically stated otherwise, the term “Fab” relates to a polypeptide excluding the Fc portion of the antibody.
  • the Fab may be conjugated to a polypeptide comprising other components, such as further antigen binding domains, costimulatory domains, linkers, peptide spacers, transmembrane domains, endodomains, and accessory proteins.
  • Fab polypeptides can be generated using conventional techniques known in the art and are well-described in the literature.
  • An Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • the term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are included.
  • Antigen-binding peptide scaffolds such as complementarity-determining regions (CDRs) are used to generate protein-binding molecules in accordance with the embodiments.
  • CDRs complementarity-determining regions
  • a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and/or be easy to produce, express, and purify. Skerra, J Mol Recognit, 13: 167-87 (2000).
  • the protein scaffolds can be sourced from, but not limited to: fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratri copeptide repeat”.
  • Such proteins are described in US Patent Publication Nos. 2010/0285564, 2006/0058510, 2006/0088908, 2005/0106660, and PCT Publication No. W02006/056464, each of which are specifically incorporated herein by reference in their entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal NO synthase (PIN) may also be used.
  • PIN neuronal NO synthase
  • selective binding agent refers to a molecule that binds to an antigen.
  • Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab 1 , F(ab')2, single chain antibodies, peptides, peptide fragments and proteins
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • immunologically reactive means that the selective binding agent or antibody of interest will bind with antigens present in a biological sample.
  • immuno complex refers the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.
  • affinity refers the strength with which an antibody or selective binding agent binds an epitope. In antibody binding reactions, this is expressed as the affinity constant (Ka or ka sometimes referred to as the association constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of the antibody to its antigen relative to the binding strength of the antibody to an unrelated amino acid sequence. Affinity can be expressed as, for example, 20- fold greater binding ability of the antibody to its antigen then to an unrelated amino acid sequence.
  • vidity refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • immunosorbent and “preferentially binds” are used interchangeably herein with respect to antibodies and/or selective binding agent.
  • examples of some experimental methods that can be used to determine the KD value are: enzyme-linked immunosorbent assays (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE).
  • ELISA enzyme-linked immunosorbent assays
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • ACE affinity capillary electrophoresis
  • Antibodies deemed useful in certain embodiments may have an affinity constant (Ka) of about, at least about, or at most about 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M or any range derivable therein.
  • antibodies may have a dissociation constant of about, at least about or at most about 10' 6 , 10' 7 , 10" s , 10' 9 , 10' 10 M, or any range derivable therein. These values are reported for antibodies discussed herein and the same assay may be used to evaluate the binding properties of such antibodies.
  • the epitope of an antigen is the specific region of the antigen for which an antibody has binding affinity.
  • the epitope is the specific residues (or specified amino acids or protein segment) that the antibody binds with high affinity.
  • An antibody does not necessarily contact every residue within the protein. Nor does every single amino acid substitution or deletion within a protein necessarily affect binding affinity.
  • epitope and antigenic determinant are used interchangeably to refer to the site on an antigen to which B and/or T cells respond or recognize
  • Polypeptide epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide.
  • An epitope typically includes at least 3, and typically 5-10 amino acids in aunique spatial conformation.
  • Epitope specificity of an antibody can be determined in a variety of ways.
  • One approach involves testing a collection of overlapping peptides of 15 amino acids spanning the full sequence of the protein and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids).
  • the peptides are immobilized in separate wells of a microtiter dish. Immobilization can be accomplished, for example, by biotinylating one terminus of the peptides. This process may affect the antibody affinity for the epitope, therefore different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for the purposes of comparison. This is useful for identifying end-specific antibodies.
  • additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments. An antibody or antigen-binding fragment is screened for binding to each of the various peptides.
  • the epitope is defined as a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding.
  • the antibodies of the present invention may be modified, such that they are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the epitopes of the present invention.
  • Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.
  • amino acid sequences of antibodies or antigen-binding regions thereof are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% sequence identity.
  • conservative amino acid replacements are contemplated.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families based on the chemical nature of the side chain; e.g., acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine).
  • acidic aspartate, glutamate
  • basic lysine, arginine, histidine
  • nonpolar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • uncharged polar glycine, asparagine, glutamine, cysteine, serine, thre
  • Standard ELISA, Surface Plasmon Resonance (SPR), or other antibody binding assays can be performed by one skilled in the art to make a quantitative comparison of antigen binging affinity between the unmodified antibody and any polypeptide derivatives with conservative substitutions generated through any of several methods available to one skilled in the art.
  • fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function.
  • the antigen-binding domain may be multi-specific or multivalent by multimerizing the antigen-binding domain with VH and VL region pairs that bind either the same antigen (multi-valent) or a different antigen (multi-specific).
  • glycosylation variants of antibodies wherein the number and/or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide.
  • Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861).
  • antibody protein variants comprise a greater or a lesser number of N- linked glycosylation sites than the native antibody.
  • N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline.
  • the substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain.
  • substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide.
  • the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid
  • one or more new N-linked glycosylation sites are created.
  • Antibodies typically have an N-linked glycosylation site in the Fc region.
  • Additional antibody variants include cysteine variants, wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g , serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines
  • the polypeptides can be pegylated to increase biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the polypeptide.
  • PEG polyethylene glycol
  • Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the invention to obtain PEGylated derivatives of antibodies. See, e g., EP 0 154 316 and EP 0 401 384.
  • the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant.
  • TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, and polyvinyl alcohols.
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins.
  • the derivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment.
  • the derivatized antibody can comprise, for example, a detectable (or labeling) moiety (e g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e g., administration to a subject, such as a human subject, or other in vivo or in vitro uses).
  • a detectable (or labeling) moiety e g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead
  • an antibody or an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
  • polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule. See, e g., EP 0322094 and EP 0 486 525.
  • the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen.
  • the polypeptides may also be conjugated to a therapeutic agent to provide a therapy in combination with the therapeutic effect of the polypeptide.
  • Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as a siRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences.
  • RNase ribonuclease
  • DNase I DNase I
  • an antisense nucleic acid an inhibitory RNA molecule
  • an inhibitory RNA molecule such as a siRNA molecule
  • an immunostimulatory nucleic acid aptamers
  • ribozymes triplex forming molecules
  • the functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules
  • antibodies and antibody-like molecules that are linked to at least one agent to form an antibody conjugate or payload.
  • it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like.
  • a reporter molecule is defined as any moiety that may be detected using an assay.
  • Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands.
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to be detected, and/or further quantified if desired.
  • detectable labels include, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like.
  • labels are, but not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and a- or p-galactosidase.
  • Antibody conjugates include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme to generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase.
  • Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds.
  • the uses of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
  • Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).
  • contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active tox
  • the immunoconjugate can be provided in the form of a fusion protein.
  • an antibody may be conjugated to various therapeutic substances in order to target the cell surface antigen.
  • conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens
  • an antibody is conjugated to one or more drug moieties (D) through a linker (L).
  • the ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody.
  • Antibody drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug.
  • a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • the antibody may be conjugated to a “receptor” (such as streptavidin) for utilization in tumor or cancer cell pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e g., avidin) which is conjugated to a cytotoxic agent (e g , a radionucleotide)
  • a receptor such as streptavidin
  • a ligand e g., avidin
  • cytotoxic agent e g , a radionucleotide
  • Examples of an antibody-drug conjugates known to a person skilled in the art are pro-drugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605- 614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26: 151-172 (1997); U.S. Pat. No. 4,975,278).
  • ADC include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide.
  • the conjugated peptide may be a heterologous signal (or leader) polypeptide, e g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His).
  • Antibody-containing fusion proteins may comprise peptides added to facilitate purification or identification of the antibody (e g., poly- His).
  • An antibody polypeptide also can be linked to the FLAG® (Sigma-Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio/Technology 6: 1204 (1988), and U.S. Pat. No. 5,011,912.
  • Oligomers that contain one or more antibody polypeptides may be employed as antagonists. Oligomers may be in the form of covalently linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibody polypeptides are contemplated for use. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.
  • oligomers comprise multiple antibody polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the antibody polypeptides.
  • Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization.
  • Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of antibody polypeptides attached thereto, as described in more detail below.
  • Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3 -6 -diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bos(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyl
  • derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated.
  • Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).
  • Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., 1987).
  • polypeptides, peptides, and proteins and immunogenic fragments thereof for use in various embodiments can also be synthesized in solution or on a solid support in accordance with conventional techniques. See, for example, Stewart and Young, (1984); Tam et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
  • a polyclonal antibody is prepared by immunizing an animal with an antigen or a portion thereof and collecting antisera from that immunized animal.
  • the antigen may be altered compared to an antigen sequence found in nature.
  • a variant or altered antigenic peptide or polypeptide is employed to generate antibodies.
  • Inocula are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent to form an aqueous composition.
  • Antisera is subsequently collected by methods known in the arts, and the serum may be used as-is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual 1988).
  • Myeloma cell lines suited for use in hybridoma- producing fusion procedures preferably are non-antibody-producing and have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas).
  • the fusion partner includes a property that allows selection of the resulting hybridomas using specific media.
  • fusion partners can be hypoxanthine/aminopterin/thymidine (HAT)-sensitive.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • hybridomas selection of hybridomas can be performed by culturing the cells by singleclone dilution in microtiter plates, followed by testing the individual clonal supernatants (after two to three weeks) for the desired reactivity. Fusion procedures for making hybridomas, immunization protocols, and techniques for isolation of immunized splenocytes for fusion are known in the art.
  • SLAM selected lymphocyte antibody method
  • Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation, or overall functional properties relative to being a treatment for infection. Thus, monoclonal antibodies may have alterations in the amino acid sequence of CDRs, including insertions, deletions, or substitutions with a conserved or non-conserved amino acid
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants that may be used in accordance with embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • Exemplary adjuvants may include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and/or aluminum hydroxide adjuvant.
  • BRM biologic response modifiers
  • Cimetidine CIM; 1200 mg/d
  • CYP Cyclophosphamide
  • cytokines such as -interferon, IL-2, or IL- 12, or genes encoding proteins involved in immune helper functions, such as B-7.
  • a phage-display system can be used to expand antibody molecule populations in vitro.
  • human antibodies may be produced in a non-human transgenic animal, e.g., a transgenic mouse capable of producing multiple isotypes of human antibodies to protein (e g., IgG, IgA, and/or IgE) by undergoing V-D-J recombination and isotype switching.
  • a non-human transgenic animal e.g., a transgenic mouse capable of producing multiple isotypes of human antibodies to protein (e g., IgG, IgA, and/or IgE) by undergoing V-D-J recombination and isotype switching.
  • this aspect applies to antibodies, antibody fragments, and pharmaceutical compositions thereof, but also non-human transgenic animals, B-cells, host cells, and hybridomas that produce monoclonal antibodies.
  • Applications of human antibodies include, but are not limited to, detect a cell expressing an anticipated protein, either in vivo or in vitro, pharmaceutical preparations containing the antibodies of the present invention, and methods of treating disorders by administering
  • Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • Antigens for this purpose typically have six or more contiguous amino acids, and optionally are conjugated to a carrier, such as a hapten
  • a carrier such as a hapten
  • transgenic animals are produced by incapacitating the endogenous mouse immunoglobulin loci encoding the mouse heavy and light immunoglobulin chains therein, and inserting into the mouse genome large fragments of human genome DNA containing loci that encode human heavy and light chain proteins. Partially modified animals, which have less than the full complement of human immunoglobulin loci, are then crossbred to obtain an animal having all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for the immunogen but have human rather than murine amino acid sequences, including the variable regions. For further details of such methods, see, for example, International Patent Application Publication Nos.
  • mice described above contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and K chain loci (Lonberg et al., Nature 368:856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or K chains and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG K monoclonal antibodies (Lonberg et al., supra; Lonberg and Huszar, Intern. Ref. Immunol.
  • HuMAb mice The preparation of HuMAb mice is described in detail in Taylor et al., Nucl. Acids Res. 20:6287-6295 (1992); Chen et al., Int. Immunol. 5:647-656 (1993); Tuaillon et al., J. Immunol. 152:2912-2920 (1994); Lonberg et al., supra; Lonberg, Handbook of Exp. Pharmacol. 113:49-101 (1994); Taylor et al., Int. Immunol. 6:579-591 (1994); Lonberg and Huszar, Intern. Ref.
  • WO 93/1227; WO 92/22646; and WO 92/03918 the disclosures of all of which are hereby incorporated by reference in their entirety for all purposes.
  • Technologies utilized for producing human antibodies in these transgenic mice are disclosed also in WO 98/24893, and Mendez et aL, Nat. Genetics 15: 146-156 (1997), which are herein incorporated by reference.
  • the HCo7 and HCol2 transgenic mice strains can be used to generate human antibodies.
  • antigen-specific humanized monoclonal antibodies with the desired specificity can be produced and selected from the transgenic mice such as those described above. Such antibodies may be cloned and expressed using a suitable vector and host cell, or the antibodies can be harvested from cultured hybridoma cells. Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom et al., J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol. 222:581 (1991)). One such technique is described in International Patent Application Publication No. WO 99/10494 (herein incorporated by reference), which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk-receptors using such an approach. C. Antibody Fragments Production
  • Antibody fragments that retain the ability to recognize the antigen of interest will also find use herein.
  • a number of antibody fragments are known in the art that comprise antigen-binding sites capable of exhibiting immunological binding properties of an intact antibody molecule and can be subsequently modified by methods known in the arts.
  • Functional fragments including only the variable regions of the heavy and light chains, can also be produced using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. See, e.g., Inbar et al., Proc. Nat. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem. 19:4091-4096 (1980).
  • Single-chain variable fragments may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH).
  • scFvs can form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., Prot. Eng. 10:423 (1997); Kort et al., Biomol. Eng 18:95-108 (2001)).
  • VL- and VH-comprising polypeptides By combining different VL- and VH-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., Biomol Eng 18:31-40 (2001)) Antigen-binding fragments are typically produced by recombinant DNA methods known to those skilled in the art.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single chain polypeptide (known as single chain Fv (sFv or scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci.
  • sFv or scFv single chain Fv
  • Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility. Antigen-binding fragments are screened for utility in the same manner as intact antibodies.
  • Such fragments include those obtained by amino-terminal and/or carboxy-terminal deletions, where the remaining amino acid sequence is substantially identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full- length cDNA sequence.
  • Antibodies may also be generated using peptide analogs of the epitopic determinants disclosed herein, which may consist of non-peptide compounds having properties analogous to those of the template peptide These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30: 1229 (1987).
  • ABSiPs antibody like binding peptidomimetics
  • These analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidiner, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference in their entirety for any purpose.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Such compounds are often developed with the aid of computerized molecular modeling.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used in certain embodiments of the invention to generate more stable proteins.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide
  • a phage display library can be used to improve the immunological binding affinity of the Fab molecules using known techniques. See, e.g., Figini et al., J. Mol. Biol. 239:68 (1994).
  • the coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used. in.
  • Obtaining Encoded Antibodies there are nucleic acid molecule encoding antibody polypeptides (e g., heavy or light chain, variable domain only, or full-length). These may be generated by methods known in the art, e g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules.
  • the nucleic acid molecules may be used to express large quantities of recombinant antibodies or to produce chimeric antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and other antibody derivatives. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization.
  • contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains)
  • Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigenbinding portion thereof.
  • expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • DNAs encoding partial or full-length light and heavy chains are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences.
  • expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences.
  • flanking sequences typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secretion
  • ribosome binding site a sequence encoding a leader sequence for polypeptide secretion
  • polyadenylation sequence a polylinker region for inserting the nucleic acid encoding the polypeptid
  • Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides.
  • Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.
  • nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art.
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S.
  • Patent 5,789,215 incorporated herein by reference
  • electroporation U.S. Patent No. 5,384,253, incorporated herein by reference
  • calcium phosphate precipitation Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990
  • DEAE dextran followed by polyethylene glycol
  • direct sonic loading Fechheimer et al., 1987
  • liposome mediated transfection Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al , 1989; Kato et al., 1991
  • microprojectile bombardment PCT Application Nos.
  • Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.
  • contemplated are the use of host cells into which a recombinant expression vector has been introduced.
  • Antibodies can be expressed in a variety of cell types.
  • An expression construct encoding an antibody can be transfected into cells according to a variety of methods known in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • the antibody expression construct can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NFAT- 1 or NF -KB, both of which are transcription factors that can be activated upon T-cell activation.
  • Control of antibody expression allows T cells, such as tumor- targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells.
  • T cells such as tumor- targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells.
  • T cells such as tumor- targeting T cells
  • cytokine signaling both in the T cells themselves and in surrounding endogenous immune cells.
  • One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector.
  • techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded
  • a selectable marker e g., for resistance to antibiotics
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts
  • nucleic acid molecule encoding either or both of the entire heavy and light chains of an antibody or the variable regions thereof may be obtained from any source that produces antibodies. Methods of isolating mRNA encoding an antibody are well known in the art. See e.g., Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, e.g., Kabat et al., 1991, supra. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed in a cell into which they have been introduced and the antibody isolated.
  • aspects of the present disclosure relate to treatment, analysis, or use of a virus.
  • methods for treatment or prevention of a viral infection In some embodiments, disclosed are compositions comprising one or more anti-viral agents.
  • methods for diagnosis of a viral infection In some embodiments, disclosed are methods for detection of a virus in a sample.
  • the virus is from the family Coronaviridae.
  • Coronaviridae is a family of enveloped, positive-sense, single-stranded RNA viruses.
  • Coronavirus is the common name for Coronaviridae and Orthocoronavirinae (also referred to as Coronavirinae).
  • the family Coronaviridae is organized in 2 sub-families, 5 genera, 23 sub-genera and approximately 40 species. They are enveloped viruses having a positive-sense single-stranded RNA genome and a nucleocapsid having helical symmetry.
  • the genome size of coronaviruses ranges from approximately 26-32 kilobases.
  • the present disclosure encompasses treatment or prevention of infection of any virus in the Coronaviridae family.
  • the disclosure encompasses treatment or prevention of infection of any virus in the subfamily Coronavirinae and including the four genera, Alpha-, Beta-, Gamma-, and Deltacoronavirus.
  • the disclosure encompasses treatment or prevention of infection of any virus in the genus of Betacoronavirus, including the subgenus Sarbecovirus and including the species of severe acute respiratory syndrome-related coronavirus.
  • the disclosure encompasses treatment or prevention of infection of any virus in the species of severe acute respiratory syndrome-related coronavirus, including the strains severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes COVID-19).
  • the disclosure encompasses treatment or prevention of infection any isolate, strain, type (including Type A, Type B and Type C; Forster et al., 2020, PNAS, available on the World Wide Web at doi.org/10.1073/pnas.2004999117), cluster, or sub-cluster of the species of severe acute respiratory syndrome-related coronavirus, including at least SARS-CoV-2.
  • the virus has a genome length between 29000 to 30000, between 29100 and 29900, between 29200 and 29900, between 29300 and 29900, between 29400 and 29900, between 29500 and 29900, between 29600 and 29900, between 29700 and 29900, between 29800 and 29900, or between 29780 and 29900 base pairs in length.
  • SARS-CoV-2 viruses include the following listed in the NCBI GenBank® Database, and these GenBank® Accession sequences are incorporated by reference herein in their entirety: (a) LC534419 andLC534418 and LC528233 and LC529905 (examples of different strains from Japan); (b) MT281577 and MT226610 and NC_045512 and MN996531 and MN908947 (examples of different strains from China); (c) MT281530 (Iran); (d) MT126808 (Brazil); (e) MT020781 (Finland); (f) MT093571 (Sweden); (g) MT263074 (Peru); (h) MT292582 and MT292581 and MT292580 and MT292579 (examples of different strains from Spain); (i) examples from the United States, such as MT276331 (TX); MT276330 (FL
  • the disclosure encompasses treatment or prevention of infection of any of these or similar viruses, including viruses whose genome has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
  • the disclosure encompasses treatment or prevention of infection of any of these or similar viruses, including viruses whose genome has its entire sequence that is greater than 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
  • the present disclosure includes methods of treatment or prevention of infection of a virus having a genome sequence represented by GenBank® Accession No.
  • NC 045512 origin Wuhan, China and any virus having a genome sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identity to a genome sequence represented by GenBank® Accession No. NC 045512.
  • SARS-CoV-2 proteins are described in detail in, for example, Yoshimoto F. K.
  • a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues.
  • wild-type refers to the endogenous version of a molecule that occurs naturally in an organism.
  • wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to generate an immune response.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.
  • polypeptide also includes and antibody fragment described herein as well as antibody domains, such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFRW1, HFRW2, HFRW3, HFRW4, LFRW1, LFRW2, LFRW3, LFRW4, VH, VL, CH, or CL.
  • a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed.
  • the protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods.
  • SPPS solid-phase peptide synthesis
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • an antibody, antigen binding fragment, protein or polypeptide may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,
  • polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).
  • domain refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.
  • the antibody, antigen binding fragment, polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
  • the antibody, antigen binding fragment, protein, or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • the antibody, antigen binding fragment, or polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • the antibody, antigen binding fragment, protein, or polypeptide may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • nucleic acid molecule there is a nucleic acid molecule, antibody, antigen binding fragment, protein, or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • a polypeptide (e.g., antibody, antibody fragment, Fab, etc.) of the disclosure comprises a CDR that is at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) in sequence to one of SEQ ID NOS: 1-2804.
  • a polypeptide comprises 1, 2, and/or 3 CDRs from one of SEQ ID NOS: 1-2804.
  • the CDR may be one that has been determined by Kabat, IMGT, or Chothia.
  • a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes (e g., addition of 1 or 2 amino acids, deletions of 1 or 2 amino acids, substitution) with respect to these 1, 2, or 3 CDRs.
  • a polypeptide comprises additionally or alternatively, an amino acid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to the amino acid sequence of the variable region that is not a CDR sequence, i.e., the variable region framework.
  • the CDRs are CDR1, CDR2, and CDR3.
  • a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes (e g., addition of 1 or 2 amino acids, deletions of 1 or 2 amino acids, substitution) with respect to CDR1 , CDR2, or CDR3.
  • the CDRs of SEQ ID NOS : 1-2804 may further comprise 1, 2, 3, 4, 5, or 6 additional amino acids at the amino or carboxy terminus of the CDR,
  • the additional amino acids may be from the heavy and/or light chain framework regions of SEQ ID NOS:44-76, that are shown as immediately adjacent to the CDRs.
  • embodiments relate to polypeptides comprising anHCDRl (i.e., CDR-H1), HCDR2(i e., CDR- H2), HCDR3(i.e., CDR-H3), LCDRl(i.e., CDR-L1), LCDR2(i.e., CDR-L2), and/or LCDR3(i.e., CDR-L3) with at least or at most or exactly 1, 2, 3, 4, 5, 6 or 7 amino acids at the amino end of the CDR or at the carboxy end of the CDR, wherein the additional amino acids are the 1, 2, 3, 4, 5, 6, or 7 amino acids of Table 1 or SEQ ID NOS: 1-2804 that are shown as immediately adjacent to the CDRs.
  • antibodies comprising one or more CDRs, wherein the CDR is a fragment of Table 1 or SEQ ID NOS: 1-2804 and wherein the fragment lacks 1, 2, 3, 4, or 5 amino acids from the amino or carboxy end of the CDR.
  • the CDR may lack one, 2, 3, 4, 5, 6, or 7 amino acids from the carboxy end and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the framework region of the amino end of the CDR.
  • the CDR may lack one, 2, 3, 4, 5, 6, or 7 amino acids from the amino end and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the framework region of the carboxy end of the CDR.
  • an antibody may be alternatively or additionally humanized in regions outside the CDR(s) and/or variable region(s).
  • a polypeptide comprises additionally or alternatively, an amino acid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to the amino acid sequence of the variable region that is not a CDR sequence, i.e., the variable region framework.
  • a polypeptide or protein comprises 1, 2, 3, 4, 5, or 6 CDRs from either or both of the light and heavy variable regions of Table 1 or SEQ ID NOS: 1-2804, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3 amino acid changes with respect to these CDRs.
  • parts or all of the antibody sequence outside the variable region have been humanized.
  • a protein may comprise one or more polypeptides.
  • a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide.
  • nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases.
  • Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org).
  • Genbank and GenPept databases on the World Wide Web at ncbi.nlm.nih.gov/
  • the Universal Protein Resource UniProt; on the World Wide Web at uniprot.org.
  • the coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • compositions of the disclosure there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
  • concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 8, 0.9, 1.0, 1.5, 2 0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6 0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • amino acid subunits of a protein may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.
  • Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants.
  • a variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type.
  • a variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein.
  • a variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.
  • Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.
  • Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which
  • substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.
  • hydropathy index of amino acids may be considered.
  • the hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain.
  • Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics.
  • the importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3 0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4), proline (-0.5+1); alanine ( _ 0.5); histidine (— 0.5); cysteine (-1.0), methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 are included, in other embodiments, those which are within ⁇ 1 are included, and in still other embodiments, those within ⁇ 0.5 are included.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue.
  • amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions may be made in the naturally occurring sequence.
  • substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts.
  • conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).
  • nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding peptides and polypeptides of the disclosure, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein.
  • nucleic acids encoding fusion proteins that include these peptides are also provided.
  • the nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).
  • polynucleotide refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences.
  • Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.
  • the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • a nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.
  • polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • nucleic acid segments regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably
  • nucleic acids can be any length.
  • nucleic acid fragments of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
  • hybridization The nucleic acids that hybridize to other nucleic acids under particular hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, e g , Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6 3.1-6.3 6. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5* sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6* SSC, and a hybridization temperature of 55° C.
  • a stringent hybridization condition hybridizes in 6> ⁇ SSC at 45° C , followed by one or more washes in 0. l x SSC, 0.2% SDS at 68° C.
  • nucleic acids comprising nucleotide sequence that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other typically remain hybridized to each other.
  • Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigenic peptide or polypeptide) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site- directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol However it is made, a mutant polypeptide can be expressed and screened for a desired property.
  • a polypeptide e.g., an antigenic peptide or polypeptide
  • Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues.
  • one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes See, eg., Romain Studer et al., Biochem. J. 449:581-594 (2013).
  • the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity Examples of qualitative changes include altering the antigen specificity of an antibody.
  • nucleic acid molecules are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences.
  • a nucleic acid molecule can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of a given polypeptide.
  • the nucleic acid molecules may be used as probes or PCR primers for specific nucleic acid sequences
  • a nucleic acid molecule probe may be used in diagnostic methods or a nucleic acid molecule PCR primer may be used to amplify regions of DNA that could be used, inter alia, to isolate nucleic acid sequences for use in producing the engineered cells of the disclosure.
  • the nucleic acid molecules are oligonucleotides.
  • Probes based on the desired sequence of a nucleic acid can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of interest.
  • the probe can comprise a label group, e g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide.
  • nucleic acid molecule encoding polypeptides, antibodies, or antigen binding fragments of the disclosure.
  • the nucleic acid molecules may be used to express large quantities of polypeptides. If the nucleic acid molecules are derived from a nonhuman, non-transgenic animal, the nucleic acid molecules may be used for humanization of the antibody or TCR genes.
  • contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e g , a fragment containing one or more CDRs or one or more variable region domains).
  • Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigenbinding portion thereof.
  • expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody heavy and/or light chain, antibody fragments, and probes thereof.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • DNAs encoding the polypeptides or peptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences.
  • expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secreti
  • Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides.
  • Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.
  • nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art.
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S.
  • Patent 5,789,215 incorporated herein by reference
  • electroporation U.S. Patent No. 5,384,253, incorporated herein by reference
  • calcium phosphate precipitation Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990
  • DEAE dextran followed by polyethylene glycol
  • direct sonic loading Fechheimer et aL, 1987
  • liposome mediated transfection Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991
  • microprojectile bombardment PCT Application Nos.
  • Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.
  • the present disclosure includes methods for treating disease and modulating immune responses in a subject in need thereof.
  • the disclosure includes cells that may be in the form of a pharmaceutical composition that can be used to induce or modify an immune response
  • compositions according to the current disclosure will typically be via any common route. This includes, but is not limited to parenteral, orthotopic, intradermal, subcutaneous, orally, transdermally, intramuscular, intraperitoneal, intraperitoneally, intraorbitally, by implantation, by inhalation, intraventricularly, intranasally or intravenous injection.
  • compositions of the present disclosure e g., compositions comprising SARS-CoV-2 protein-binding polypeptides
  • compositions and therapies of the disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.
  • the manner of application may be varied widely. Any of the conventional methods for administration of pharmaceutical compositions comprising cellular components are applicable.
  • the dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subj ect.
  • administrations of at most or at least 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the administrations may range from 2-day to 12-week intervals, more usually from one to two week intervals.
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.
  • the pharmaceutical compositions of the current disclosure are pharmaceutically acceptable compositions.
  • compositions of the disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions and the preparations can also be emulsified.
  • Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Sterile injectable solutions are prepared by incorporating the active ingredients (e.g., polypeptides of the disclosure) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by fdtered sterilization.
  • active ingredients e.g., polypeptides of the disclosure
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • An effective amount of a composition is determined based on the intended goal.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subj ect, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • compositions and related methods of the present disclosure may also be used in combination with the administration of additional therapies such as the additional therapeutics described herein or in combination with other traditional therapeutics known in the art.
  • compositions and treatments disclosed herein may precede, be cocurrent with and/or follow another treatment or agent by intervals ranging from minutes to weeks.
  • agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic agents would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one may contact the cell, tissue or organism with two, three, four or more agents or treatments substantially simultaneously (i.e., within less than about a minute).
  • one or more therapeutic agents or treatments may be administered or provided within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day,
  • the treatments may include various “unit doses.”
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition.
  • the quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • the quantity to be administered depends on the treatment effect desired.
  • An effective dose is understood to refer to an amount necessary to achieve a particular effect In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents.
  • doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein.
  • doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the therapeutically effective or sufficient amount of the immune checkpoint inhibitor, such as an antibody and/or microbial modulator, that is administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations.
  • the therapy used is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example.
  • a therapy described herein is administered to a subject at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles.
  • the dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of this therapy is easily monitored by conventional techniques.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 LIM to 150 ⁇ M.
  • the effective dose provides a blood level of about 4 ⁇ M to 100 ⁇ M.; or about 1 ⁇ M to 100 ⁇ M; or about 1 ⁇ M to 50 ⁇ M; or about 1 ⁇ M to 40 ⁇ M; or about 1 ⁇ M to 30 ⁇ M; or about 1 ⁇ M to 20 ⁇ M; or about 1 ⁇ M to 10 ⁇ M; or about 10 ⁇ M to 150 ⁇ M; or about 10 ⁇ M to 100 ⁇ M; or about 10 ⁇ M to 50 ⁇ M; or about 25 ⁇ M to 150 ⁇ M; or about 25 ⁇ M to 100 ⁇ M; or about 25 ⁇ M to 50 ⁇ M; or about 50 ⁇ M to 150 ⁇ M; or about 50 ⁇ M to 100 ⁇ M (or any range derivable therein).
  • the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subj ect: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 27, 28,
  • the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of ⁇ g/ml or mM (blood levels), such as 4 ⁇ M to 100 u ⁇ 1. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
  • polypeptides can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like.
  • a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like.
  • Such labeled polypeptides can be used for diagnostic techniques, either in vivo, or in an isolated test sample or in methods described herein.
  • label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition.
  • the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • the labels can be suitable for small scale detection or more suitable for high-throughput screening.
  • suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes
  • the label may be simply detected or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
  • Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).
  • luminescent probes include, but are not limited to, aequorin and luciferases.
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red.
  • suitable optical dyes are described in the Haugland, Richard P (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker.
  • Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens/polypeptides, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavidin.
  • haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten polypeptides. See, Harlow and Lane (1988) supra.
  • methods involve obtaining or evaluating a sample from a subject.
  • the sample may include a sample obtained from any source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva.
  • any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing.
  • the biological sample can be obtained without the assistance of a medical professional.
  • a sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject.
  • the biological sample may be a heterogeneous or homogeneous population of cells or tissues.
  • the biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein.
  • the sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.
  • the sample may be obtained by methods known in the art.
  • the samples are obtained by biopsy.
  • the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art.
  • the sample may be obtained, stored, or transported using components of a kit of the present methods.
  • multiple samples such as multiple esophageal samples may be obtained for diagnosis by the methods described herein.
  • multiple samples such as one or more samples from one tissue type (for example esophagus) and one or more samples from another specimen (for example serum) may be obtained for diagnosis by the methods.
  • multiple samples such as one or more samples from one tissue type (e.g.
  • samples from another specimen may be obtained at the same or different times. Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.
  • the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist.
  • the medical professional may indicate the appropriate test or assay to perform on the sample.
  • a molecular profiling business may consult on which assays or tests are most appropriately indicated.
  • the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.
  • the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy.
  • the method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy.
  • multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.
  • the sample is a fine needle aspirate of a esophageal or a suspected esophageal tumor or neoplasm.
  • the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.
  • the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party.
  • the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business.
  • the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.
  • a medical professional need not be involved in the initial diagnosis or sample acquisition.
  • An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit.
  • OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit.
  • molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately.
  • a sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.
  • the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist.
  • the specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample.
  • the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample.
  • the subject may provide the sample.
  • a molecular profiling business may obtain the sample.
  • the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include both freshly isolated cells and ex vivo cultured, activated or expanded cells. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses.
  • a host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell
  • a transformed cell includes the primary subject cell and its progeny.
  • transfection can be carried out on any prokaryotic or eukaryotic cell.
  • electroporation involves transfection of a human cell.
  • electroporation involves transfection of an animal cell.
  • transfection involves transfection of a cell line or a hybrid cell type.
  • the cell or cells being transfected are cancer cells, tumor cells or immortalized cells.
  • tumor, cancer, immortalized cells or cell lines are induced and in other instances tumor, cancer, immortalized cells or cell lines enter their respective state or condition naturally.
  • the cells or cell lines can be A549, B-cells, B16, BHK-21, C2C12, C6, CaCo-2, CAP/, CAP-T, CHO, CHO2, CHO-DG44, CH0-K1, COS-1, Cos-7, CV-1, Dendritic cells, DLD-1, Embryonic Stem (ES) Cell or derivative, H1299, HEK, 293, 293 T, 293FT, Hep G2, Hematopoietic Stem Cells, HOS, Huh-7, Induced Pluripotent Stem (iPS) Cell or derivative, lurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, Mesenchymal Cells, Min-6, Monocytic cell, Neuro2a, NIH 3T3, NIH3T3L1, K562, NK-cells, NS0, Panc-1, PC12, PC-3, Peripheral blood cells, Plasma cells, Primary Fibroblasts,
  • kits containing compositions of the disclosure or compositions to implement methods of the disclosure
  • kits can be used to detect the presence of a SARS-CoV-2 virus in a sample.
  • a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein.
  • a kit contains one or more polypeptides capable of binding to a SARS-CoV-2 spike protein, including polypeptides disclosed herein.
  • a kit may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Fabs disclosed herein for detecting a SARS-CoV-2 spike protein.
  • a kit comprises a detection pair.
  • a kit comprises an enzyme.
  • a kit comprises a substrate for an enzyme.
  • Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.
  • Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.
  • Kits for using probes, synthetic nucleic acids, nonsynthetic nucleic acids, and/or inhibitors of the disclosure for prognostic or diagnostic applications are included as part of the disclosure.
  • negative and/or positive control nucleic acids, probes, and inhibitors are included in some kit embodiments.
  • Kits may further comprise instructions for use.
  • a kit comprises instructions for detecting a SARS-CoV-2 virus in a sample.
  • Example 1 Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2
  • Serum antibodies and MBCs have potential to act as the first line of defense against SAR.S-CoV-2 infection 11 15 ' 17 .
  • the inventors collected peripheral blood mononuclear cells (PBMCs) and serum from 25 subjects between April and May of 2020 upon recovery from SAR.S-CoV-2 viral infection (Extended Data Table 1 and Extended Data Table 2).
  • PBMCs peripheral blood mononuclear cells
  • the inventors generated probes to bait-sort enriched B cells for subsequent single cell RNA sequencing analysis by conjugating distinct phycoerythrin (PE)-streptavidin (SA)-oligos to individual biotinylated antigens (FIG. la).
  • PE phycoerythrin
  • SA streptavidin
  • the inventors detected small percentages (0.02-0.26%) of SARS-CoV-2-reactive total CD19 + B cells, which were subsequently used to prepare 5’ transcriptome, immunoglobulin (Ig) VDJ, and antigen- specific probe feature libraries for sequencing (FIG. la, b).
  • the inventors detected increased percentages of antigen-specific B cells within the memory B cell (MBC) compartment FIG. lb, CD19 + CD27 + CD38 int ), though the inventors sorted on total CD19 + antigen-specific B cells to ensure adequate coverage of all potential reactive B cells and to optimize sequence library preparation and downstream analysis as the antigen-specific population was rare.
  • the inventors integrated data from 17 subjects with high-quality sequencing results using Seurat to remove batch effects and identified 12 transcriptionally distinct B cell clusters based on transcriptional expression profiles (FIG. 1c). It was immediately evident that B cells specific to the spike, NP, ORF7a, and ORF8 were found amongst multiple B cell subsets, with spike-specific B cells substantially enriched in clusters 4, 5, 7, and 9 (FIG. Id, e) Analysis of Ig isotypes and degree of Ig variable heavy chain somatic hypermutations (VH SHM) suggested that clusters 0-2, 8, 10, and 11 represented naive- or innate-like B cell clusters predominantly composed of IgM and IgD B cells.
  • VH SHM variable heavy chain somatic hypermutations
  • clusters 3, 4, 5, 6, 7, 9, and 12 strongly indicated B cell subsets more similar to MBCs or plasma cells, as they exhibited a higher degree of class switch recombination (CSR) and/or increased numbers of VH SHM (FIG If).
  • CSR class switch recombination
  • VH SHM VH SHM
  • the inventors next addressed whether the probe intensities generated from the feature libraries correlated with antigen-specific reactivity by plotting intensities for distinct probes against one another to observe true specificity (cells that fall directly onto the x or y axis) vs. non-specific binding (cells that fall on the diagonal).
  • the inventors observed hundreds of cells specific to the spike, ORF8, NP, and to a lesser degree, ORF7a (FIG. 1g). For clusters 0, 1, 2, and 8, the inventors observed that the majority of cells were not uniquely specific for any one probe, and instead tended to bind more than one probe in a polyreactive or non-specific manner, consistent with innate-like B cells 18 .
  • clusters 4, 5, 6, 7, and 9 exhibited highly specific binding toward the spike, NP, and ORF8, with the majority targeting the spike (FIG. 6c).
  • the data suggest the B cell response to SARS-CoV-2 is comprised of multiple functionally distinct B cell subsets enriched for binding to distinct viral targets.
  • Clusters 3 and 5 appeared to be specific IgM memory subsets (FIG. If, FIG. 6c), while clusters 4, 7, 9, and 12 displayed high specificity, CSR, and SHM, demonstrating an affinity -matured memory phenotype (FIG. If, FIG. 6c). As naive B cells and MBCs are quiescent, clusters 4, 5, 7, and 9 were similar to cluster 2 in pseudotime analysis (FIG. 2a-b) 19 . Lastly, cluster 6 was of interest as these cells displayed the greatest frequency of SHM and IgA CSR, and may have arisen in the context of a mucosal immune response.
  • cluster 7 which has recently been shown to be involved in MBC differentiation in mice (FIG. 7) 20 .
  • cluster 12 appeared to be LLPCs or precursors thereof by expression of genes associated with LLPC fate, including prdml, xbpl, and mctnf (FIG. 7) 19,21,22
  • FIG. 1 the antigen-specific probe data (FIG. 1), these results confirm that clusters representing classical MBCs are enriched for spike binding while B cells targeting internal proteins are enriched in activated naive and innate-like B cell subsets.
  • B cells targeting immunogenic targets such as ORF8 and NP compared to the spike are unknown.
  • the inventors further analyzed isotype frequencies, VH SHM, VH gene usages, and frequencies of B cells against these targets within distinct B cell subsets.
  • the majority of antigen-specific B cells were of the IgM isotype with a limited degree of CSR
  • Spike-specific B cells were primarily enriched in MBC and LLPC-like clusters 4, 5, 7, 9, and 12 while NP- and ORF8-specific B cells were largely found within naive- and innate-like clusters but also within MBC clusters (FIG. 3a-l).
  • HC heavy chain
  • LC light chain
  • FIG. 8a-b the inventors did not observe differences in heavy chain (HC) or light chain (LC) complementarity determining region 3 length by antigen targeting
  • HC and LC isoelectric points (pl) for spike-reactive B cells were generally lower than NP- or ORF8-reactive B cells (FIG. 8c-d), and LC SHM was greater for spike-reactive B cells (FIG. 8e).
  • VH-24, VH3-7, and VH3- 9 were the highest represented VH gene usages exclusively associated with non-RBD spike reactivity, and VH1-24 usage was enriched in cluster 7, an MBC-like cluster (FIG. 3m-n, FIG. 6b).
  • the inventors addressed the neutralization ability of all synthesized mAbs using a live virus plaque assay and determined that all mAbs cloned against NP and ORF8 were non-neutralizing, while mAbs against the RBD and other epitopes of the spike were largely neutralizing at varying degrees of potency (FIG. 4c-d). As anti-spike mAbs were predominantly neutralizing and enriched in memory, these MBC subsets may serve as a biomarker for superior immunity to SARS-CoV-2.
  • the inventors next analyzed the distribution of B cell subsets and frequencies of B cells specific to the spike, NP, ORF7a, and ORF8 in sets of patients stratified by age, sex, and duration of symptoms from the single cell dataset.
  • the inventors normalized antigen probe signals by a centered log-ratio transformation individually for each subject; all B cells were clustered into multiple probe hit groups according to their normalized probe signals, and cells that were negative to all probes or positive to all probes (non-specific) were excluded from the analysis.
  • the inventors identified substantial variation in antigen targeting amongst individual subjects (FIG. 5c).
  • this study highlights the diversity of B cell subsets expanded upon novel infection with SARS-CoV-2 Using this approach, the inventors identified that B cells against the spike, ORF8, and NP differ in their ability to neutralize, derive from functionally distinct and differentially adapted B cell subsets, and correlate with clinical parameters such as age, sex, and symptom duration.
  • the B cell clusters herein may provide biomarkers in the form of distinct B cell populations that can be used to evaluate future responses to various vaccine formulations.
  • the identification of LLPC precursors in the blood following infection and vaccination has been long sought after, as they serve as a bonafide marker of long-lived immunity 24,25 . Future studies elucidating distinct identities and functions of these subsets are necessary and will provide key insights into B cell immunology.
  • T cell responses to SARS-CoV-2 ORF proteins are prevalent in convalescent COVID-19 patients, and recent studies suggest impaired T cell responses in aged COVID- 19 patients impact antibody responses 10 ’ 29 ’ 30 ’ 42 . More research is warranted to definitively determine whether B cell targeting of distinct SARS-CoV- 2 antigens correlates with age and disease severity. Addressing these questions will be critical for determining correlates of protection and developing a vaccine capable of protecting the most vulnerable populations.
  • Lymphocytes were purified by Lymphoprep Ficoll gradient (Thermo Fisher) and contaminating red blood cells were lysed by ACK buffer (Thermo Fisher). Cells were frozen in Fetal Bovine Serum (FBS, Gibco) with 10% Dimethyl sulfoxide (DMSO, Sigma) prior to downstream analysis On the day of sorting, B cells were enriched using the human pan B cell EasySepTM enrichment kit (STEMCELL).
  • FBS Fetal Bovine Serum
  • DMSO Dimethyl sulfoxide
  • Soluble recombinant proteins were produced as described previously 33 .
  • inclusion body proteins were washed, denatured, reduced, and then renatured by rapid dilution following standard methods 34 .
  • the refolding buffer consisted of 400 mM arginine, 100 mM Tris-HCl, 2 mM EDTA, 200 ⁇ M ABESF, 5 mM reduced glutathione, and 500 ⁇ M oxidized glutathione at a final pH of 8.3.
  • the soluble-refolded protein was collected over a 10 kDa ultrafiltration disc (EMD Millipore, PLGC07610) in a stirred cell concentrator and subjected to chromatography on a HiLoad 26/60 Superdex S75 column (GE Healthcare).
  • Site-specific biotinylation with BirA enzyme was done following the manufacture's protocol (Avidity) except that the reaction buffer consisted of lOOmM Tris-HCl (pH 7.5) 150 mM NaCl, with 5 mM MgC12 in place of 0.5 M Bicine at pH 8.3. Unreacted biotin was removed by passage through a 7K MWCO desalting column (Zeba spin, Thermo Fisher).
  • Full-length SARS-CoV- 2 NP was cloned into pET21a with a hexahistidine tag and expressed using BL21(DE3)-RIL E. coli in Terrific Broth (bioWORLD). Following overnight induction at 25°C, cells were lysed in 20 mM Tris-HCl pH 8.5, 1 M NaCl, 5 mM ⁇ -mercaptoethanol, and 5 mM imidazole for nickel-affinity purification and size exclusion chromatography.
  • Biotinylated proteins were then conjugated to Biolegend TotalSeqTM PE streptavidin- (PE-SA) oligos at a 0.72:1 molar ratio of antigen to PE-SA
  • the amount of antigen was chosen based on a fixed amount of 0.5 pg PE-SA and diluted in a final volume of 10 pL.
  • PE-SA was then added gradually to 10 pl biotinylated proteins 5 times on ice, 1 pl PE-SA (0.1 mg/ml stock) every 20 minutes for a total of 5 pl (0.5 pg) PE-SA.
  • the reaction was then quenched with 5 pl 4mM PierceTM biotin (Thermo Fisher) for 30 minutes for a total probe volume of 20 pL. Probes were then used immediately for staining
  • PBMCs were thawed and B cells were enriched using EasySepTM pan B cell magnetic enrichment kit (STEMCELL).
  • B cells were stained with a panel containing CD 19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences).
  • B cells were stained with surface stain master mix and each CO VID-19 antigen probe for 30 minutes on ice in IX PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin.
  • Cells were stained with probe at a 1 : 100 dilution (NP, ORF7a, ORF8, RBD) or 1 :200 dilution (spike) Cells were subsequently washed with IX PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in IX PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in IX PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19 + /antigen- PE + were sorted as probe positive. The PE + gate was drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.
  • VDJ, 5’, and probe feature libraries were prepared using the 1 OX Chromium System (10X Genomics, Pleasanton, CA). The Chromium Single Cell 5’ Library and Gel Bead v2 Kit, Human B Cell V(D)J Enrichment Kit, and Feature Barcode Library Kit were used. All steps were followed as listed in the manufacturer’s instructions. Specifically, user guide CG000186 Rev D was used. Final libraries were pooled and sequenced using the NextSeq550 (Illumina, San Diego, CA) with 26 cycles apportioned for read 1, 8 cycles for the i7 index, and 134 cycles for read 2.
  • NextSeq550 Illumina, San Diego, CA
  • Seurat version 3.2.0, an R package, for transcriptome, cell surface protein and antigen probe analysis
  • IgBlast version 1.15, for immunoglobulin gene analysis
  • Seurat was used for cell quality control, data normalization, data scaling, dimension reduction (both linear and non-linear), clustering, differential expression analysis, batch effects correction, and data visualization. Unwanted cells were removed according to the number of detectable genes (number of genes ⁇ 200 or >2500 were removed) and percentage of mitochondrial genes for each cell.
  • a soft threshold of percentage of mitochondrial genes was set to the 95 th percentile of the current dataset distribution, and the soft threshold was subject to a sealing point of 10% as the maximum threshold in the case of particularly poor cell quality.
  • Transcriptome data were normalized by a log-transform function with a scaling factor of 10,000 whereas cell surface protein and antigen probe were normalized by a centered log-ratio (CLR) normalization.
  • CLR log-ratio
  • the inventors used variable genes in principal component analysis (PCA) and used the top 15 principal components (PCs) in non-linear dimension reduction and clustering. High-quality cells were then clustered by Louvain algorithm implemented in Seurat under the resolution of 0 6. Differentially expressed genes for each cell cluster were identified using a Wilcoxon rank-sum test implemented in Seurat. Batch effects correction analysis was performed using an Anchor method implemented in Seurat to remove batch effects across different datasets. All computational analyses were performed in R (version 3.6.3).
  • Trajectory analyses were performed using Monocle 3 (version 0.2.2) 35,36 , Seurat 3, and the SeuratWrappers package (version 0.2.0) 37 .
  • Cells from multiple subjects were integrated to remove batch effects using Seurat, and all cells were clustered into two non-connected partitions.
  • the inventors then performed trajectory analysis on the main partition containing the majority of the cells and clusters (clusters 0-11). Pseudotime analysis of cells was also inferred from this major partition using Monocle3.
  • the root node of the pseudotime analysis was set to cluster 2, a naive B cell subset with the lowest degree of VH gene SHM and CSR.
  • Representative antibodies from each subject were chosen for synthesis by choosing random samplings of B cells that bound to a given antigen probe with higher intensity relative to all other probes.
  • B cells with varying ranges of probe-binding intensities were chosen for confirmation by ELISA
  • B cells binding to all probes in a polyreactive manner were also chosen and validated for polyreactivity by polyreactivity ELISA (see methods below).
  • Immunoglobulin heavy and light chain genes were obtained by 10X Genomics VDJ sequencing analysis and monoclonal antibodies (mAbs) were synthesized by Integrated DNA Technologies. Cloning, transfection, and mAb purification have been previously described 38 . Briefly, sequences were cloned into human IgGl expression vectors using Gibson assembly, and heavy and light genes were co-transfected into 293T cells (Thermo Fisher). Secreted mAbs were then purified from the supernatant using protein A agarose beads (Thermo Fisher).
  • High-protein binding microtiter plates (Costar) were coated with recombinant SARS-CoV-2 proteins at 2 ⁇ g/ml in IX PBS overnight at 4°C. Plates were washed the next morning with IX PBS 0.05% Tween and blocked with IX PBS containing 20% fetal bovine serum (FBS) for 1 hour at 37°C. Antibodies were then serially diluted 1 :3 starting at 10 ⁇ g/ml and incubated for 1 hour at 37°C.
  • HRP horseradish peroxidase
  • MBC stimulations were performed on PBMCs collected from subjects in the convalescent cohort.
  • 1 ⁇ 10 6 PBMCs were stimulated with 10 ng/ml Lectin Pokeweed Mitogen (Sigma-Aldrich), 1/100,000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), and 6 ⁇ g/ml CpG (Invitrogen) in complete RPMI in an incubator at 37°C/5% CO 2 for 5 days.
  • ELISpot white polystyrene plates (Thermo Fisher) coated with 4 ⁇ g/ml of SARS-CoV-2 spike that were blocked with 200 pl of complete RPMI. ELISpot plates were incubated with cells for 16 hours overnight in an incubator at 37°C/5% CO2. After the overnight incubation, plates were washed and incubated with anti-IgG-biotin and/or anti- IgA-biotin (Mabtech) for 2 hours at room temperature. After secondary antibody incubation, plates were washed and incubated with streptavidin-alkaline phosphatase (Southern Biotech) for 2 hours at room temperature.
  • the SARS-CoV-2/UW-001/Human/2020AVisconsin (UW-001) virus was isolated from a mild case in February 2020 and used to assess neutralization ability of mAbs.
  • Virus (-500 plaque-forming units) was incubated with each mAb at a final concentration of 10 ⁇ g/ml. After a 30-minute incubation at 37°C, the virus/antibody mixture was used to inoculate Vero E6/TMPRSS2 cells seeded a day prior at 200,000 cells per well of a TCI 2 plate. After 30 minutes at 37°C, cells were washed 3 times to remove any unbound virus, and media containing antibody (10 ⁇ g/ml) was added back to each well. 2 days after inoculation, cell culture supernatant was harvested and stored at -80°C until needed. A non-relevant Ebola virus GP mAb and PBS were used as controls.
  • a standard plaque-forming assay was performed. Confluent Vero E6/TMPRSS2 cells in a TC12 plate were infected with supernatant (undiluted, 10-fold dilutions from 10 1 to 10 -5 ) for 30 minutes at 37°C. After the incubation, cells were washed 3 times to remove unbound virus and 1.0% methylcellulose media was added over the cells. After an incubation of 3 days at 37°C, the cells were fixed and stained with crystal violet solution in order to count the number plaques at each dilution and determine virus concentration given as plaque-forming units (PFU)/ml. A stringent cutoff for neutralization was chosen as 100-fold greater neutralization relative to the negative control mAb. MAbs were screened once for neutralization.
  • PFU plaque-forming units
  • SARS-CoV nucleocapsid protein antagonizes IFN- beta response by targeting initial step of IFN-beta induction pathway, and its C-terminal region is critical for the antagonism.
  • Example 2 Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non- neutralizing viral targets
  • MCCs memory B cells
  • SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convales- cent patients several months post symptom onset.
  • NP nucleoprotein
  • ORF8 open reading frame 8
  • the inventors characterized the SARS-CoV-2- specific B cell repertoire in 38 COVID-19 patients, both severe acute and convalescent, approximately 1.5-4.5 months post-symptom onset, using oligo-tagged antigen bait sorting and single-cell RNA sequencing (RNA-seq).
  • RNA-seq single-cell RNA sequencing
  • RNA-seq Single-cell RNA-seq reveals substantial complexity among endemic HCoV- and SARS-CoV-2-specific B cells MBCs have potential to act as an early line of defense against viral infection, as they rapidly expand into antibody-secreting cells (ASCs) upon antigen reencounter.
  • ASCs antibody-secreting cells
  • the inventors collected peripheral blood mononuclear cells (PBMCs) and serum between April and May 2020 from 10 severely infected acute subjects and 28 subjects upon recovery from SARS-CoV-2 viral infection (Tables SI- S4).
  • PBMCs peripheral blood mononuclear cells
  • SARS-CoV-2 SARS- CoV-2
  • spike RBD spike RBD
  • NP NP
  • ORF8 SARS- CoV-2
  • SA PE- streptavidin
  • Fig. 10a BioLegend TotalSeq
  • the inventors included an empty PE-SA-oligo, along with hantavirus PUUV, an irrelevant viral antigen control, on APC.
  • the inventors included a cocktail of spike proteins from four coronavirus strains that cause mild upper respiratory infections in the vast majority of individuals: HCoV-229E, HCoV-NL63, HCoV-HKUl, and HCoV-OC43, on one additional APC-SA-oligo.
  • the inventors then integrated sequencing re- suits from all 38 subjects using Seurat to remove batch effects and identified 16 transcriptionally distinct B cell clusters on the basis of expression profiles (Fig. 10b).
  • Adopting the ROGUE scoring method which compares how similar all transcriptomes within a cluster are to one another, the inventors determined that most clusters were highly pure, with the majority having a score over 0.9 (1.0 indicating 100% purity) (Fig. 10c; Liu et al., 2020).
  • the inventors ensured that the feature libraries correlated with singleprobe antigen-specific reactivity using a series of filtering steps to re- move cells that were probe negative, multi -reactive and non- specific, empty PE-SA + , or Hanta-PUUV + .
  • the inventors did not identify obvious differences in B cell subset dis- tribution or antigen reactivity in B cells from severe acute sub- jects analyzed early (days 0, 1, and 3) or late (days 7 and 14 post-convalescent plasma therapy (Figs. 16c-d). In summary, this method revealed substantial complexity in the B cell response to distinct coronavirus antigens, which the inventors then further dissected by subset.
  • the SARS-CoV-2-specific B cell landscape is defined by naive-like and MBC subsets
  • VH variable heavy chain somatic hyper- mutation
  • SHM somatic hyper- mutation
  • Fig. l ib Clusters 0, 1, 3, and 5 ex- pressed Ig genes with little to no SHM or CSR and gene signatures associated with naive B cells, suggesting that these subsets were composed of naive-like B cells or very recently activated B cells (Figs. 1 la-b).
  • clusters with patterns of higher CSR and SHM were further investigated for memory gene signatures.
  • clusters 4, 6, 7, and 8 as MBCs; clusters 2, 9, and 13 as recent memory or GC emigrants; clusters 10, 11, and 15 as ASCs; and clusters 12 and 14 as innate-like in nature, though genes for these subsets are not well defined in humans (Figs a-b, bottom).
  • the inventors generated scores for each cluster and projected them onto UMAP, allowing us to visualize how closely associated clusters relate to one another on the basis of their B cell subset score (Fig. 11c).
  • the inventors further visualized how cells clustered on the basis of identity by overlaying key gene signatures for MBCs, recent GC emigrants, and ASCs (Table S6). Some cells were outside of their home cluster, suggesting that they were in the course of differen- tiation and highlighting the plasticity of cells in an active immune response (Figs. 17a-c).
  • ASC clusters 10, 11, and 15 dis- played a high degree of SHM, suggesting that they may derive from preexisting memory that was driven against endemic HCoV spike proteins (Fig. I la).
  • SARS-CoV-2-specific B cells display unique repertoire features and protective ability
  • VK variable light-chain kappa
  • VL variable light-chain lambda
  • a B cell was considered non-RBD spike-specific if it bound full-length spike probe and not RBD probe, and a cell that bound both RBD and full-length spike was considered to be RBD-specific.
  • the inventors found that B cells against HCoV spike, non-SARS2 RBD spike epitopes, and the SARS2 RBD were enriched for VH1-69 gene usage (Figs. 13a-c).
  • VH1-69 is commonly used by broadly neutralizing antibodies against the hemagglutinin stalk domain of influenza vi- ruses, as well as the gp120 co-receptor binding site of HIV-1, because of its ability to bind conserved hydrophobic regions of viral envelope glycoproteins (Chen et al., 2019).
  • VH1-69 usage by B cells that cross-react to SARS-CoV-2 and HCoV has also been indicated (Wee et al., 2020).
  • VH1-69 usage for B cells targeting HCoV spike and SARS2 spike non-RBD epitopes was predominantly enriched in convalescent visit 1 subjects and not convalescent visit 2, suggesting that the repertoire may continue to evolve months after infection (Figs. 13a-b, right).
  • VH gene usages were enriched in both convales- cent visits, regardless of antigen specificity.
  • VH3-7 and VH1-24 were also commonly used, which the inventors confirmed by characterizing cloned mAbs from the cohort (Fig.
  • NP-specific B cells used similar variable gene usages as RBD-specific B cells (Fig. 13d)
  • ORF8-specific B cells were enriched for VH1-2 and VH1-3 paired with VK3-20, and enrichment for these VH genes persisted across both convalescent time points (Fig. 13e).
  • Fig. 13f the frequency of the top ten heavy and light chain gene pairings (total antigen-specific cells) shared across subjects for both convalescent time points.
  • the inventors next inves- tigated the binding, neutralization potency, and in vivo protec- five ability of mAbs cloned from select BCRs. To do so, the inventors expressed nearly 100 mAbs against the SARS2 spike, NP, and ORF8 from convalescent subjects, representing a multi- tude of clusters (Table S7). Cells from which to clone antibodies were chosen at random and were not chosen on the basis of specific sequence features. However, the inventors note that the results described herein may be affected by sampling bias, as only a small subset of antigen-specific mAbs were cloned.
  • NP- and ORF8-specific mAbs were entirely non-neutralizing (Fig. 14b).
  • Figs 14c-d animal models of SARS-CoV-2 infection, the inventors confirmed that anti- RBD antibodies were therapeutically protective in vivo, preventing weight loss and reducing lung viral titers relative to PBS control and an irrelevant Ebola anti-GP133 mAb (Figs 14c-d).
  • mAbs to NP and ORF8 were non-neutralizing in vitro, they might still provide protection in vivo, potentially through Fc- mediated pathways if the proteins were exposed on the virus or cell surface at appreciable levels.
  • B cell immunodominance is shaped by age, sex, and disease severity
  • Serum antibody titers to the spike and intracellular proteins are shown to correlate with age, sex, and SARS-CoV-2 severity (Atyeo et al., 2020; Guthmiller et al., 2021; Robbiani et al., 2020).
  • the inventors there- fore analyzed the distribution of B cell subsets and frequencies of B cells specific to the spike, NP, and ORF8 in convalescent subjects stratified by age, sex, and severity of disease. Disease severity was stratified into three categories: mild, moderate, and severe, on the basis of symptom duration and symptoms experienced (Ta- ble SI), as defined previously (Guthmiller et al., 2021).
  • Fig. 15a The inventors found that reactivity of total B cells toward different antigens varied widely by subject, likely reflecting host-intrinsic differences.
  • Fig. 15b With age, the inventors identified a decrease in the generation of spike-specific B cells and an increase in ORF8 and NP-specific B cells (Fig. 15b).
  • Fig. 15c the percentage of total spike-specific B cells was reduced in subjects with more severe disease, whereas ORF8-specific B cells were increased. 15c).
  • Fig. 15c the inventors identi- fied that women had increased percentages of ORF8- reactive cells, whereas men showed slightly greater percentages of NP- reactive cells.
  • this study highlights the diversity of B cell subsets expanded upon novel infection with SARS-CoV-2.
  • the inventors identified that B cells against the spike, ORF8, and NP differ in their ability to neutralize and derive from functionally distinct and differentially adapted B cell subsets; that MBC output over time shifts from the spike to intracellular anti- gens; and that targeting of these antigens is affected by age, sex, and disease severity
  • the COVID-19 pandemic continues to pose one of the greatest public health and policy challenges in modem history, and robust data on long-term immunity are critically needed to evaluate future decisions regarding CO VID-19 responses.
  • This approach combined three powerful aspects of B cell biology to address human immunity to SARS-CoV-2: B cell transcriptome, Ig sequencing, and recombinant mAb characterization.
  • the inventors show that antibodies targeting key protective spike epitopes are en- riched within MBC populations, but over time the MBC pool con- tinues to adapt toward non-protective intracellular antigens, which could be a molecular hallmark of waning B-cell-mediated protection. This is further evidence that widespread vaccination, which only elicits a response to the spike, may be critical to end the pandemic.
  • HCoV spike bind- ing B cells adapt to the SARS2 spike and can provide protection is of interest for the potential generation of a universal coronavi- rus vaccine. Further investigation into the protection afforded by cross-reactive antibodies is warranted, as previous studies have identified cross- reactive HCoV and SARS1 binding antibodies can neutralize SARS-CoV-2 (Ng et al., 2020; Wee et al., 2020). Vaccine-induced responses to the spike will also be shaped by preexisting immunity and should be investigated.
  • SARS2 spike-specific B cells from the convalescent cohort were enriched in memory
  • the inventors also identified MBCs and ASCs to HCoV spike, which waned 4.5 months after infection. This later time point coincided with an increase in overall numbers and percentage of ORF8- and NP-specific MBCs, which displayed a marked increase in SHM. This phenotype was pronounced in older patients, who exhibited reduced MBC targeting of the spike. Patients who were older, were fe- male, and had more severe disease showed increased B cell targeting of ORF8, and older patients tended to generate more memory to intracellular proteins over time. The inventors identified B cells targeting these intracellular proteins as exclusively non-neutralizing and non-protective.
  • these ob- servations may be explained by reduced adaptability of B cells or increased reliance on CD4 T cell help forB cell activation, which have been observed in aged individuals upon viral infec- tions and are dysregulated in aged patients (Dugan et al., 2020b; Henry et al., 2019) Furthermore, T cell responses to SARS-CoV-2 intracellular proteins are prevalent in convales- cent COVID-19 patients (Grifoni et al., 2020; Le Bert et al., 2020; Peng et al., 2020).
  • the shift in memory output during convalescence may also reflect the massive difference in pro tein availability, with each virion producing only dozens of spikes but thousands of intracellular proteins (Grifoni et al., 2020; Lu et al., 2021; Yao et al., 2020).
  • PBMCs were collected from leukoreduction filters or blood draws within 2 hours post-collection and, if applicable, flushed from the filters using sterile IX Phosphate-Buffered Saline (PBS, GIBCO) supplemented with 0.2% Bovine Serum Albumin (BSA, Sigma).
  • PBS sterile IX Phosphate-Buffered Saline
  • BSA Bovine Serum Albumin
  • Lymphocytes were purified by Lymphoprep Ficoll gradient (Thermo Fisher) and contaminating red blood cells were lysed by ACK buffer (Thermo Fisher). Cells were frozen in Fetal Bovine Serum (FBS, GIBCO) with 10% Dimethyl sulfoxide (DMSO, Sigma) prior to downstream analysis. On the day of sorting, B cells were enriched using the human pan B cell EasySepTM enrichment kit (STEMCELL).
  • FBS Fetal Bovine Serum
  • DMSO Dimethyl sulfoxide
  • SARS-CoV-2 and Hanta PUUV proteins were obtained from the Krammer laboratory at Mt. Sinai, the Joachimiak laboratory at Ar- gonne, and the Fremont laboratory at Washington University.
  • pCAGGS expression constructs for the spike protein, spike RBD, and hanta PUUV were obtained from the Krammer lab at Mt. Sinai and produced in house in Expi293F suspension cells (Thermo Fisher). Sequences for the spike and RBD proteins as well as details regarding their expression and purification have been previously described (Amanat et al., 2020; Stadlbauer et al., 2020).
  • Proteins were biotinylated for 2 hours on ice using EZ- Link Sulfo-NHS-Biotin, No-Weigh Format (Thermo Fisher) according to the manufacturer’s instructions, unless previously Avi-tagged and biotinylated (ORF8 protein, Fremont laboratory). Truncated cDNAs encoding the Ig-like domains of ORF8 were inserted into the bacterial expression vector pET-21(a) in frame with a biotin ligase recognition sequence at the c-terminus (GLNDIFEAQKIEWHE). Soluble recombinant proteins were produced as described previously (Nelson et al., 2005).
  • inclusion body proteins were washed, denatured, reduced, and then renatured by rapid dilution following standard methods (Nelson et al., 2014).
  • the refolding buffer consisted of 400 mM arginine, 100 mM Tris-HCl, 2 mM EDTA, 200 mM ABESF, 5 mM reduced glutathione, and 500 mM oxidized glutathione at a final pH of 8.3.
  • the soluble-refolded protein was collected over a 10 kDa ultrafiltration disc (EMD Millipore, PLGC07610) in a stirred cell concentrator and subjected to chromatography on a HiLoad 26/60 Superdex S75 column (GE Health- care).
  • Site specific biotinylation with BirA enzyme was done following the manufacture’s protocol (Avidity) except that the reaction buffer consisted of 1 OOmM Tris-HCl (pH7.5) 150 mM NaCl, with 5mM MgC12 in place of 0.5 M Bicine at pH 8.3. Unreacted biotin was removed by passage through a 7K MWCO desalting column (Zeba spin, Thermo Fisher).
  • Full-length SARS-CoV-2 NP was cloned into pET21a with a hexahistidine tag and expressed using BL21(DE3)-RIL E. coli in Terrific Broth (bioWORLD) Following overnight indue- tion at 25°C, cells were lysed in 20 mM Tris-HCl pH 8.5, 1 M NaCl, 5 mM b-mercaptopethanol, and 5 mM imidazole for nickel- affmity purification and size exclusion chromatography.
  • Endemic HCoV spike proteins (HCoV-229E, HCoV-NL63, HCoV-HKUl, and HCoV-OC43) were purchased from Sino Biological.
  • Biotinylated proteins were then conjugated to Biolegend TotalSeq PE streptavidin (PE- SA), APC streptavidin (APC-SA), or non-fluorescent streptavidin (NF-SA) oligos at a 0.72:1 molar ratio of antigen to PE-SA, APC-SA, or NF-SA.
  • the amount of antigen was chosen based on a fixed amount of 0.5 mg PE-SA, APC-SA, or NF-SA and diluted in a final volume of 10 mL.
  • PE-SA, APC-SA, or NF-SA was then added gradually to 10 mL biotinylated proteins 5 times on ice, 1 mL PE-SA, APC-SA, or NF-SA (0.1 mg/ml stock) every 20 minutes for a total of 5 mL (0.5 mg) PE-SA, APC-SA, or NF-SA.
  • the reaction was then quenched with 5 mL 4mM Pierce biotin (Thermo Fisher) for 30 minutes for a total probe volume of 20 mL. Probes were then used immediately for staining.
  • B cells were thawed and B cells were enriched using EasySepTM pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD 19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosci- ences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each CO VID-19 antigen probe for 30 minutes on ice in IX PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin.
  • Cells were stained with probe at a 1 : 100 dilution (NP, ORF8, RBD, PUUV, empty PE-SA) or 1 :200 dilution (spike, endemic HCoV spikes). Cells were subsequently washed with IX PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in IX PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in IX PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi).
  • Cells that were viable/CD19 + /antigen-PE + or viable/CD19 + /antigen-APC + were sorted as probe positive.
  • the PE + and APC + gates were drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.
  • VDJ, 5°, and probe feature libraries were prepared using the 1 OX Chromium System (10X Genomics). The Chromium Single Cell 5° Library and Gel Bead v2 Kit, Human B Cell V(D)J Enrichment Kit, and Feature Barcode Library Kit were used. All steps were followed as listed in the manufacturer’s instructions. Specifically, user guide CG000186 Rev D was used. Severe acute infected samples were pooled post-sort and hashtagged (Biolegend), and run as a single sample, to account for low cell numbers Final libraries were pooled and sequenced using the NextSeq550 (Illumina) with 26 cycles apportioned for read 1, 8 cycles for the i7 index, and 134 cycles for read 2.
  • NextSeq550 Illumina
  • the inventors adopted Cell Ranger (version 3.0.2) for raw sequencing processing, including 5° gene expression analysis, antigen probe anal- ysis, and immunoprofiling analysis of B cells. Based on Cell Ranger output, the inventors performed downstream analysis using Seurat (version 3.9.9, an R package, for transcriptome, cell surface protein and antigen probe analysis) and IgBlast (version 1.15, for immunoglobulin gene analysis). For transcriptome analysis, Seurat was used for cell quality control, data normalization, data scaling, dimension reduction (both linear and non-linear), clustering, differential expression analysis, batch effects correction, and data visualization.
  • Seurat version 3.9.9, an R package, for transcriptome, cell surface protein and antigen probe analysis
  • IgBlast version 1.15, for immunoglobulin gene analysis
  • Seurat was used for cell quality control, data normalization, data scaling, dimension reduction (both linear and non-linear), clustering, differential expression analysis, batch effects correction, and data visualization.
  • Un- wanted cells were removed according to the number of detectable genes (number of genes ⁇ 200 or > 2500 were removed) and per- centage of mitochondrial genes for each cell.
  • a soft threshold of percentage of mitochondrial genes was set to the 95 th percentile of the current dataset distribution, and the soft threshold was subject to a sealing point of 10% as the maximum threshold in the case of particularly poor cell quality.
  • Transcriptome data were normalized by a log-transform function with a scaling factor of 10,000 whereas cell surface protein and antigen probe were normalized by a centered log-ratio (CLR) normalization.
  • CLR log-ratio
  • PCA prin- cipal component analysis
  • PCs principal components
  • ROGUE scoring an entropy-based metric for assessing the purity of single cell populations, adapted from a previous study (Liu et al., 2020).
  • the expression entropy for each gene was calculated using “SE fun” from the “ROGUE” package (version 1.0). Based on the expression entropy, the ROGUE score for each cluster was calculated using the “rogue” function from the same package with parameters “platform’ ’ set to ‘ ‘UMI’ ’ and “span’ ’ set to 0.6.
  • Antigen probe signals were normalized by a centered log-ratio transformation individually for each subject. All B cells were sub- sequently clustered into multiple probespecific groups according to their normalized probe signals. By investigating all normal- ized antigen-probe binding signals, the inventors arbitrarily set a threshold equal to 1 for all normalized probe signals to distinguish probe binding cells as “positive” or “negative.” Cells that were negative to all probes were clustered into the “negative” group; those positive to only one probe were clustered into corresponding probe-specific groups; and those that were positive to multiple probes were further investigated.
  • the naive score was calculated based on the genes BACH2, ZBTB16, APBB2, SPRY1, TCL1A, and IKZF2, the MBC score was calculated based on the genes CD27, CD86, RASSF6, TOX, TRERF1, TRPV3, POU2AF1, RORA, TNFRSF13B, CD80, and FCRL5, the ASC score was calculated based on genes PRDM1 , MANF, XBP1, IL6R, BCL6, IRF4, TNFRSF17, and CD38 and the GC emigrant score was calculated based on genes NT5E, MKI67, CD40, CD83, TNFRSF13B, MAP3K8, MAP3K1, and FAS.
  • Representative antibodies from each subject were chosen for synthesis by choosing random samplings of B cells that bound to a given antigen probe with higher intensity relative to all other probes. B cells with varying ranges of probe-binding intensities were chosen for confirmation by ELISAs. In addition, B cells representing select public clonal expansions were also chosen for cloning. B cells binding to all probes in a polyreactive manner were also chosen and validated for polyreactivity by polyreactivity ELISA (see methods below).
  • Genomics VDJ sequencing analysis and monoclonal antibodies were synthesized by Integrated DNA Technologies. Cloning, transfection, and mAb purification have been previously descri- bed (Guthmiller et al., 2019). Briefly, sequences were cloned into human IgGl expression vectors using Gibson assembly, and heavy and light genes were co-transfected into 293T cells (Thermo Fisher). Secreted mAbs were then purified from the supernatant using protein A agarose beads (Thermo Fisher).
  • High-protein binding microtiter plates (Costar) were coated with recombinant SARS-CoV-2 proteins at 2 mg/ml in IX PBS overnight at 4°C. Plates were washed the next morning with IX PBS 0.05% Tween and blocked with IX PBS containing 20% fetal bovine serum (FBS) for 1 hour at 37°C. Antibodies were then serially diluted 1 :3 starting at 10 mg/ml and incubated for 1 hour at 37°C.
  • HRP horseradish peroxidase
  • eBiosciences horseradish peroxidase-conjugated goat anti-human IgG antibody diluted 1 : 1000 (Jackson Immuno Research) was used to detect binding of mAbs, and plates were subsequently developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioRad). To standardize the assays, control antibodies with known binding charac- teristics were included on each plate and the plates were developed when the absorbance of the control reached 3.0 OD 405 units. All experiments were performed in duplicate 2-3 times.
  • Plates were coated with 10 mg/ml cardiolipin in 100% ethanol and allowed to dry overnight. Plates were washed with water and blocked with IX PBS/0.05%Tween/lmM EDTA. MAbs were diluted 1 mg/ml in PBS and serially diluted 4-fold, and added to plates for 1.5 hours. Goat anti-human IgG-HRP (Jackson Immunoresearch) was diluted 1 :2000 in PBS/0.05%Tween/lmM EDTA and added to plates for 1 hour.
  • the SARS-CoV-2/UW-001/Human/2020/Wisconsin (UW-001) virus was isolated from a mild case in February 2020 and used to assess neutralization ability of monoclonal antibodies (mAbs).
  • Virus (-500 plaque-forming units) was incubated with each mAb at a final concentration of 10 mg/ml. After a 30-minute incubation at 37°C, the virus/antibody mixture was used to inoculate Vero E6/ TMPRSS2 cells seeded a day prior at 200,000 cells per well of a TC12 plate. After 30 minutes at 37°C, cells were washed three times to remove any unbound virus, and media containing antibody (10 mg/ml) was added back to each well. Two days after inoculation, cell culture supernatant was harvested and stored at -80°C until needed. A non- relevant Ebola vims GP mAb and PBS were used as controls.
  • mice were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory An- imals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381—01 ). Vims inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.

Abstract

Pour répondre au besoin de l'état de la technique, les inventeurs ont complètement caractérisé le répertoire des lymphocytes B spécifiques au SARS-CoV-2 chez des patients atteints de la COVID-19 convalescents et des mAb générés contre les protéines de spicule, ORF8, et NP. Conjointement, les données des inventeurs ont révélé un aperçu clé en une distribution de sous-ensemble de spécificité antigénique et de lymphocytes B lors d'une infection à SARS-CoV-2 dans le contexte d'âge, de sexe et de gravité de maladie. Selon certains aspects, la divulgation concerne un nouvel anticorps et des fragments de liaison à l'antigène. D'autres aspects concernent des polypeptides comprenant le ou les fragments de liaison à l'antigène selon la divulgation, et des compositions comprenant les polypeptides, les anticorps et/ou les fragments de liaison à l'antigène selon la divulgation. La divulgation concerne également des acides nucléiques codant pour un anticorps ou un fragment de liaison à l'antigène selon la divulgation.
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